Vol. 11, 49–57, January 1, 2005 Clinical Cancer Research 49

Inhibitory Effect of Antagonists of Bombesin and Growth Hormone- Releasing Hormone on Orthotopic and Intraosseous Growth and Invasiveness of PC-3 Human Prostate Cancer in Nude Mice

Anton Stangelberger,1,2 Andrew V. Schally,1,2 Conclusions: Our findings show the efficacy of BN/GRP Jozsef L. Varga,1,2 Marta Zarandi,1,2 antagonists and GHRH antagonists for the treatment of advanced prostate cancer in preclinical metastatic models. Karoly Szepeshazi,1,2 Patricia Armatis,1,2 and 1,2 As BN/GRP antagonists are already in clinical trials and Gabor Halmos GHRH antagonists are effective in androgen-independent 1Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical 2 prostate cancer models, these analogues could be considered Center and Section of Experimental Medicine, Department of Medicine, for the management of advanced prostate carcinoma. Tulane University School of Medicine, New Orleans, Louisiana

ABSTRACT INTRODUCTION Purpose: To determine whether antagonists of growth Prostate cancer is the most common noncutaneous hormone-releasing hormone (GHRH) and bombesin/- malignant tumor and the second leading cause of cancer-related releasing (BN/GRP) can inhibit the orthotopic and deaths among men in the Western world (1). About 70% of all metastatic growth of PC-3 human androgen-independent newly diagnosed prostate cancers occur in men over age 65 years prostate cancers. (2). Many patients with organ-confined disease can be cured by f Experimental Design: The effects of administration of radical prostatectomy or radiation therapy, but 30% of prostate GHRH antagonist MZ-J-7-118, BN/GRP antagonist RC- cancer patients who undergo radical prostatectomy show a 3940-II, and their combination on the growth and recurrence of prostate-specific antigen elevation and some will metastatic spread of PC-3 tumors implanted orthotopically ultimately develop disseminated disease (3). Bone is the most into nude mice were evaluated. The efficacy of this common site of metastasis, other than lymph nodes, in prostate treatment on PC-3 tumors implanted intratibially and s.c. cancer patients (4, 5). Androgen deprivation, achieved by was also determined. treatment with luteinizing hormone-releasing hormone agonists Results: Treatment with MZ-J-7-118, RC-3940-II, or alone or in combination with anti-androgens, induces a remission their combination significantly inhibited the growth of PC-3 in 80% to 90% of men with advanced prostate cancer, but a tumors implanted orthotopically, intraosseously, and s.c. The relapse is observed after a median time of 12 to 33 months (6). combination of the two antagonists had the greatest effect, To date, no efficacious therapy exists for patients who inhibiting orthotopic tumor growth by 77%, intratibially relapse the first-line hormonal treatment based on androgen implanted tumors by 86%, and s.c. tumors by 86%. The deprivation. Most chemotherapeutic regimens provide essential- therapy with BN/GRP and GHRH antagonists, especially in ly only a minor benefit with respect to survival or quality of life, combination, also reduced the local tumor spread and distant although new chemotherapy combinations may provide a metastases in animals bearing orthotopic tumors. Combina- survival advantage (7, 8). Consequently, the present methods tion therapy was likewise the most effective in reducing the of treatment of relapsed prostate cancer must be improved. incidence and severity of tibial osteolytic lesions and New therapeutic approaches are being developed based on pathologic fractures in intraosseously implanted tumors. recent advances in the understanding of the role of High-affinity binding sites for BN/GRP and GHRH were and growth factors in the progression of prostate cancer (9–13). found in s.c. and orthotopic PC-3 tumor samples. MZ-J-7-118, Bombesin/gastrin-releasing (BN/GRP) are produced in RC-3940-II, and the combination of both compounds prostatic, breast, and pancreatic cancers and small cell lung inhibited in vitro growth of PC-3 cells. carcinoma and act as autocrine/paracrine growth factors (10). It has been proposed that the secretion of BN/GRP by neuroen- docrine cells might be responsible for prostate cancer progres- sion, androgen independence, and poor prognosis (12). BN/GRP Received 7/8/04; revised 9/28/04; accepted 10/14/04. was also shown to increase the proliferation and invasiveness of Grant support: Medical Research Service of the Veterans Affairs androgen-independent prostate cancer (14, 15). Specific binding Department and Zentaris GmbH (Frankfurt am Main, Germany) grant to sites for BN/GRP are present in androgen-independent cell lines Tulane University (A.V. Schally). (16) and in surgical specimens of prostate cancer (17, 18). BN/ The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked GRP antagonists, such as RC-3095 or RC-3940-II, were advertisement in accordance with 18 U.S.C. Section 1734 solely to developed for therapeutic use, in view of the tumor stimulatory indicate this fact. effect of BN/GRP and the high incidence of their receptors in Requests for reprints: Andrew V. Schally, Endocrine, Polypeptide and various human cancers (11, 17, 19). These compounds were Cancer Institute, Veterans Affairs Medical Center, 1601 Perdido Street, shown to inhibit various experimental BN/GRP receptor-positive New Orleans, LA 70112-1262. Phone: 504-589-5230; Fax: 504-566-1625; E-mail: [email protected]. cancers, including that of prostate, and are currently undergoing D2005 American Association for Cancer Research. clinical trials (9–11, 20). Antiproliferative effects of BN/GRP

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antagonists are thought to be mediated by the blockade of containing a reduced peptide bond at its COOH terminus, was mitogenic stimuli of BN/GRP and the down-regulation of also made in our laboratory as described (ref. 19; Hca is receptors for BN/GRP and epidermal growth factor (9, 11, 20). desaminophenylalanine and Tac is thiazolidine-4-carboxylic Various potent antagonistic analogues of growth hormone- acid). For daily injection, the compounds were dissolved in releasing hormone (GHRH) were also synthesized (21) for 0.1% DMSO in 10% aqueous propylene glycol solution. clinical applications in the field of cancers dependent on GHRH Cell Line and Animals. The PC-3 human androgen- and -like growth factor (IGF)-I and -II. It was shown that independent prostate cancer cell line was obtained from the GHRH antagonists inhibit a large variety of human tumors American Type Culture Collection (Manassas, VA) and main- xenografted into nude mice, including androgen-independent tained in culture as described (16). prostate cancers (9–11, 20–22). The effects of GHRH Male athymic (Ncr nu/nu) nude mice, f6 weeks old on antagonists are in part exerted indirectly through the inhibition arrival, were obtained from the National Cancer Institute- of the secretion of pituitary growth hormone and the resulting Frederick Cancer Research and Development Center (Frederick, reduction in levels of serum IGF-I, which is mainly of hepatic MD) and housed in laminar air flow cabinets under pathogen- origin (21). However, recent evidence indicates that direct free conditions with a 12-hour light/12-hour dark schedule and inhibitory effects of GHRH antagonists on the cancers are even fed autoclaved standard chow and water ad libitum. more important for tumor growth inhibition than IGF-I All experiments were done in accordance with institutional suppression (11, 23). GHRH is produced by various human guidelines of animal care. tumors, including prostate cancer, and seems to exert an In vivo Studies. Experiment 1: Orthotopic Tumor Growth. autocrine/paracrine stimulatory effect on tumors (11, 23–26). Nude mice were implanted orthotopically with PC-3 tumors to The expression of GHRH and truncated splice variants of GHRH observe the orthotopic growth and development of metastases. receptors was shown in surgical specimens from patients with Tumor tissue, harvested aseptically from donor animals after 10 locally advanced prostate cancer (26) and in LNCaP, MDA-PCa- weeks of s.c. growth, was cleaned of connective tissue and 2b, C4-2b, PC-3, and DU-145 human prostate cancer lines (22, necrotic parts, weighed, mechanically minced, suspended in 24, 25, 27). GHRH antagonists seem to act through splice variant RPMI 1640, and pushed through a medium mesh tissue sieve. receptors and decrease synthesis of IGF-I, IGF-II, and vascular The tissue suspension was centrifuged for 10 minutes at 1,000 endothelial growth factor (9, 11, 20, 22, 23, 28, 29). rpm; the supernatants were then removed and the pellets were Because metastases constitute the main cause of mortality in resuspended in RPMI 1640 at a final concentration of 500 mg prostate cancer patients, it is essential to develop treatment tumor tissue/mL. Each mouse received 0.02 mL of the regimens that inhibit effectively the growth of metastatic prostate suspension (corresponding to 10 mg of tumor tissue) injected cancer. Nude mouse models are useful for the investigation of the orthotopically with a 25-gauge needle. After 1 week, animals metastatic behavior and intraosseous growth as well as for the were randomly assigned into the following groups: control 11 assessment of the effects of antimetastatic therapies (5, 30–32). mice, GHRH antagonist MZ-J-7-118 (5 Ag/d) 8 mice, BN/GRP GHRH antagonists have been shown to inhibit the orthotopic antagonist RC-3940-II (10 Ag/d) 8 mice, and combination of MZ- growth and metastatic behavior of CAKI-1 human renal cell J-7-118 (5 Ag/d) and RC-3940-II (10 Ag/d) 8 mice. Antagonists of carcinoma and MDA-MB-435 human breast cancer models in GHRH and BN/GRP were given by daily s.c. injections. nude mice (28, 32). Controls received 100 AL/d of s.c. injected vehicle solution This study was done to investigate the effect of antagonists containing 0.1% DMSO and 10% propylene glycol in water. of BN/GRP and GHRH on the growth and metastatic spread of Treatment was started 1 week after the implantation of orthotopic human androgen-independent PC-3 prostate cancer xenografted tumors and lasted for 28 days. s.c., orthotopically, and intraosseously into nude mice. After both the first and second weeks, one animal from the control group was sacrificed by cutting the abdominal aorta under isoflurane (Abbott Laboratories, Chicago, IL) anesthesia MATERIALS AND METHODS and investigated for tumor growth and visible metastasis. The Peptides and Reagents. GHRH antagonist MZ-J-7-118 experiment was ended on day 28 when three control animals was synthesized in our laboratory by solid-phase methods died and others became moribund. Tumors or tumor compart- 1 (33), its chemical structure being [CH3-(CH2)6-CO-Tyr , ments of all animals were carefully excised, weighed, and snap 2 6 8 9 10 11 15 D-Arg , Phe(4-Cl) ,Ala,His, Tyr(Et) ,His, Abu , frozen for further investigations. All mice were investigated for 27 28 29 Nle , D-Arg , Har ]hGHRH(1-29)NH2, where Phe(4-Cl) presence and spread of tumors, with special attention to those is 4-chlorophenylalanine, Abu is a-aminobutyric acid, Nle is organs that are afflicted by prostate cancer metastases, such as norleucine, and Har is homoarginine. MZ-J-7-118 belongs to bone, liver, lung, intestines, kidneys, and lymph nodes, as well as 2 the class of D-Arg -substituted antagonistic analogues of human to occurrence of carcinosis peritonei. Liver, heart, lungs, (h) GHRH(1-29)NH2 as the earlier GHRH antagonists such as kidneys, spleen, testicles, prostate, and seminal vesicles were MZ-4-71, MZ-5-156, JV-1-36, and JV-1-38 used in previous carefully removed and weighed. Samples of metastases were oncologic and endocrinologic studies (9–11, 20–24, 28–29, checked histologically to prove the origin of prostate cancer. 32, 33). Other amino acid replacements as well as the Experiment 2: Intraosseous and s.c. Tumor Growth. PC-3 N-acyl moiety are aimed to enhance the potency of MZ-J-7- cells (5 105) from cell culture suspended in PBS were injected 118 compared with earlier antagonists. The BN/GRP antagonist with a 25-gauge needle into the medulla of the right tibia of 6 13 f 3 RC-3940-II, with the structure of [Hca ,Leu c(CH2N)- 40 nude mice. At the same time, PC-3 tumor pieces of 1mm Tac14]BN(6-14) based on the sequence of the bombesin and were implanted s.c. into the animals by trocar injection.

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Two weeks after tumor injection, when the s.c. tumors the cells. The glutaraldehyde was then removed, replaced with measured f50 mm3, the animals were divided randomly into PBS, and kept at 4jC until staining. The PBS was then three different treatment groups and one control group. Each decanted from the plates, replaced with an aqueous solution of group contained eight animals. Animals in treatment groups crystal violet (0.02% Sigma, St. Louis, MO), and incubated for received GHRH antagonist MZ-J-7-118 (5 Ag/d), BN/GRP 30 minutes. After the plates were decanted and washed to remove antagonist RC-3940-II (10 Ag/d), or a combination of the two the noninternalized stain, the crystal violet was extracted with agents. Antagonists of GHRH and BN/GRP were given by daily 70% ethanol. The absorbance at 600 nm of each well was s.c. injections. Controls received 100 AL/d of s.c. injected measured using a Beckman Coulter, Inc. (Palo Alto, CA) plate vehicle solution containing 0.1% DMSO and 10% propylene reader. Results were calculated as %T/C, where T is A600 nm of glycol in water. Treatment was started 2 weeks after the treated cultures and C is A600 nm of control cultures (medium plus implantation of intraosseous and s.c. tumors. S.c. tumor volumes vehicle). and body weights were recorded weekly. The experiment was Statistical Analysis. Data are expressed as mean F SE. ended when the animals became visibly moribund and the size of Differences between values were evaluated by one-way ANOVA the tumor in the right limb caused problems in walking. followed by Fisher-LSD multiple comparison test, P < 0.05 The animals were sacrificed on day 30 and investigated for being considered significant. tumor growth, and a complete necropsy was done. Hind limbs with macroscopically visible tumors were cut off at the proximal third of the femur, measured, and photographed. Subsequently, RESULTS the tumor-bearing legs were imaged by a RMX-625 X-ray Unit Experiment 1: Tumor Progression and Survival of (Raytheon Medical Systems, Melrose Park, IL). S.c. tumors Animals Bearing Orthotopic PC-3 Tumor Xenografts. At were excised, weighed, snap frozen, and stored at 70jC for the end of both the first and second weeks of the experiment, one receptor analysis. animal in the control group was sacrificed to observe the stages Histologic Investigation. Legs with intraosseous tumors as of tumor progression. Orthotopic PC-3 tumors grew very well as macroscopically altered tissues of bone and visceral organs aggressively. Thus, after the first week (2 weeks following were fixed in 10% neutral phosphate-buffered formalin and tumor implantation), a large tumor weighing 1,087 mg, with a investigated histologically to evaluate tumor infiltration. Legs volume of 511.82 mm3, was found in a control animal with were cross-sectioned at two to three sites, and bones were abdominal propagation and lymph node metastases. At present, decalcified overnight in Cal-EX Decalcifying Solution (Fisher no symptoms of distress could be observed. The tumor formed a Scientific, Pittsburgh, PA). The specimens were embedded in solid compartment, which involved the prostate, bladder, seminal Tissue Path Paraplast X-tra embedding medium (Fisher Scien- vesicles, and local lymph nodes as well as parts of the ileum in tific). Sections (6 Am thick) were cut and stained with H&E. contact with the primary tumor. After the second week (3 weeks Receptor Binding Assay for BN/GRP and GHRH after tumor implantation), metastatic lymph nodes in the para- Receptors. Binding characteristics of BN/GRP and GHRH aortal area and tumor spread to the colon were found. After receptors on membrane preparations from orthotopic and s.c. 3 weeks, the animals of the control group started to lose body PC-3 tumors from the control group were determined by ligand weight. One animal that died at that time had a large primary competition assays using radiolabeled Tyr4-bombesin and tumor weighing 1,754 mg, with a volume of 923.63 mm3, and GHRH antagonist JV-1-42. The preparation of tumor mem- had metastatic spread to the para-aortal lymph nodes, seminal brane fractions and receptor binding studies was described vesicles, colon, and bladder. The experiment was ended after previously (17, 26, 28). 4 weeks of treatment because of the increasing number of Receptor binding affinity (IC50) of GHRH antagonist MZ-J- moribund animals especially in the control group. In total, two 7-118 to membranes of s.c. grown PC-3 tumors from the control animals were sacrificed and three died in the control group. group was also measured in displacement experiments based on However, only one animal each died in groups treated with single the competitive inhibition of [125I]JV-1-42 binding by various agents and none in the group receiving combination therapy with concentrations (1012 to 106 mol/L)ofMZ-J-7-118as BN/GRP and GHRH antagonist. described (28). IC50 is defined as the dose of MZ-J-7-118 The body weights of some animals decreased by >20% causing 50% inhibition of the specific binding of [125I]JV-1-42. by the end of the experiment probably due to the advanced In vitro Proliferation Assay. PC-3 prostatic carcinoma tumor growth, this effect being most marked in the control cells were seeded into 96-well micro plates (Falcon, Becton group (Table 1). However, the final mean body weights of the Dickinson and Co., Lincoln Park, NJ). After a recovery period of treated groups were not significantly different from the control 24 hours, the culture medium was removed and replaced with group. serum-free medium, composed of DMEM/F-12 Ham medium Inhibition of Orthotopic Tumor Growth and Metasta- (1:1), 0.4% bovine serum albumin, and 1 mmol/L pyruvate, ses by GHRH and BN/GRP Antagonists. In mice treated with containing the test compounds dissolved in 0.1% DMSO. The RC-3940-II, MZ-J-7-118, and their combination, there was a test compounds included GHRH antagonist MZ-J-7-118, BN/ significant decrease in tumor volume and tumor weight at the GRP antagonist RC-3940-II, and their combination. Each end of the experiment compared with controls (Table 1). The treatment was done in octuplicate wells and the experiments greatest inhibition of tumor growth was in the animals that were repeated three to four times. received a combination therapy. The tumor burden was also Crystal Violet Assay. After 93 hours, the medium was significantly decreased in all treatment groups, the combination removed and 1% glutaraldehyde was added for 15 minutes to fix therapy again producing the strongest effect (Table 1).

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Table 1 Effect of BN/GRP antagonist RC-3940-II, GHRH antagonist MZ-J-7-118, and their combination on tumor growth, body weight, and survival of nude mice bearing orthotopic PC-3 androgen-independent prostate cancer Treatment groups and the Final tumor Tumor weight, Tumor burden, No. (%) of mice number of evaluable volume, mg (% inhibition) mg/g body weight with >20% animals mm3 (% inhibition) decrease in (% inhibition) body weight Control (n = 6)* 631.64 F 157.34 1,046.50 F 220.51 45.88 F 10.77 4 (67) RC-3940-II (n =7) 228.33 F 77.13 (64)y 354.63 F 113.28 (66)y 14.90 F 5.67 (68)y 0 MZ-J-7-118 (n =7) 200.88 F 82.51 (68)y 321.41 F 132.09 (69)y 18.90 F 7.15 (59)y 1 (14) Combination of both 144.25 F 50.91 (77)y 305.96 F 103.56 (71)y 11.49 F 3.82 (75)y 1 (12.5) analogues (n =8) *The control group originally had 11 animals, but one animal was sacrificed after both the first and second weeks. In addition, 1 animal died in week 3 and 2 died in week 4. yP < 0.01, versus control.

These analogues also strongly inhibited the prostate cancer three animals, indicating that the grade of tumor progression to metastases. The incidence and size of metastases were the adherent organs like seminal vesicles, bladder, or ileum was investigated at the time of necropsy. Treatment with GHRH lower. In all other groups, the tumor compartment involved the and BN/GRP antagonist did not affect the number of local prostate, bladder, seminal vesicles, and, in some cases, the metastases but reduced their sizes. The incidence and size of adherent intestines. Histologic examination showed that, in two distant metastases were remarkably reduced by all treatments. control animals, the tumor infiltration reached the fibrous Figure 1 shows a representative animal from the control group capsule of the kidney. No metastases were found in bones, liver, with extensive tumor invasion and metastases in the lower lung, or spleen, demonstrating that, after 5 weeks of orthotopic abdominal organs. Animals that received treatment had visibly growth of PC-3 tumors, only a few distant metastases occurred. less intra-abdominal spread of the tumor. The animals from the Experiment 2: Intraosseous and s.c. Implantation of control group showed the most widespread metastases in number PC-3 Xenografts. Growth of Tumors after Intraosseous Implan- and size (Table 2). All six control mice with metastatic lesions tation. Two weeks after tumor implantation, we observed a had macroscopically enlarged regional and distant lymph nodes, swelling of the hind limbs of some animals and the treatment mainly para-aortal or axillar, and metastatic lesions on the was started. After 3 weeks of treatment, animals in the control intestines. Two mice had kidney metastases. The metastases in group became visibly moribund, lost body weight, and showed the treated groups were much smaller and occurred less the first signs of staggering of their hind limb movement due to frequently. In the group treated with BN/GRP antagonist, distant tumor growth. lymph node metastases in 4 of 7 (57%) animals and intestinal One animal each died in the control, BN/GRP, and GHRH metastases in 3 of 7 (43%) animals were found. In the group that antagonist groups during the last week of treatment, but no deaths received GHRH antagonist, the tumors spread to distant lymph occurred among animals that received combined treatment. At the nodes in 5 of 7 (71%) mice but to intestines in only 2 (29%) end of the experiment, when most of the limb tumors were animals. In the group treated with the combination of GHRH and macroscopically visible (Fig. 2C and D), the volumes of tumor- BN/GRP antagonist, 3 of 8 (38%) animals had metastases in bearing hind limbs in treated groups were significantly smaller distant lymph nodes and 1 animal had metastatic involvement of compared with controls. The final tumor volumes were as follows: the ileum. The primary tumors and metastases in the combina- 576.03 F 113.97 mm3 in the control group, 221.27 F 99.70 mm3 tion group also were much smaller than in other groups, and it in the group treated with RC-3940-II (P < 0.05), 290.02 F was possible to separate the seminal vesicles and the bladder in 90.44 mm3 in the group that received MZ-J-7-118 (P < 0.05), and 86.81 F 35.75 mm3 in the combination treatment group (RC- 3940-II and MZ-J-7-118; P < 0.001), corresponding to a tumor inhibition of 62%, 50%, and 86%, respectively. We were not able to measure the tumor weight of intraosseously implanted PC-3 tumors because the tumor tissue could not be separated from bone and muscle tissue. The differences in weights between the limbs were not significant, because the legs bearing the tumor had a smaller amount of muscle than the intact legs. At the end of the experiment, the right legs of the animals were investigated by X-ray and histology. Osteolytic lesions, such as partial or complete bone dissolution and pathologic fractures, were observed in all groups. However, X-ray showed that the extent of osteolytic lesions and the number of pathologic fractures were much smaller in the animals that received the Fig. 1 Representative mouse of the control group after 35 days of combined treatment with both antagonists compared with the orthotopic growth of PC-3 human androgen-independent prostate cancer. The tumor exceeded the prostate and invaded adherent organs like control group (Fig. 2A and B). Treatment with single agents also bladder, seminal vesicles, and ileum. decreased the bone lesions but less strongly than the combined

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Table 2 Effect of BN/GRP antagonist RC-3940 II, GHRH antagonist MZ-J-7-118, and their combination on tumor spread after orthotopic implantation of PC-3 prostate cancer in nude mice: Location of the metastases and number and percentage of animals that had metastases at local and distant sites at the end of the experiment Treatment groups Total number of animals Location of metastases Local lymph nodes Distant lymph nodes Intestines Prostate/seminal vesicles Kidney Control 6 6 (100) 6 (100) 6 (100) 6 (100) 2 (33) RC-3940-II 7 6 (86) 4 (57) 3 (43) 7 (100) 0 MZ-J-7-118 7 7 (100) 5 (71) 2 (29) 7 (100) 0 Combination of both 8 8 (100) 3 (38) 1 (12.5) 8 (100) 0 analogues treatment (data not shown). Histologic analysis showed that after tumor weight of 572.29 F 164.21 mg (P < 0.05), corresponding intraosseous injection, in most cases, the tumor penetrated the to a 69% tumor inhibition. Final tumor volume and weight of the bone cortex of the tibia and infiltrated the muscles surrounding group that received MZ-J-7-118 were 565.13 F 123.77 mm3 the bones (Fig. 3A). The bones were often irregular, fragmented (P < 0.05) and 810.57 F 146.03 mg (P < 0.05), corresponding parts being mixed with newly formed trabecules (Fig. 3B). The to f56% to 58% inhibition. The strongest antiproliferative tibia was completely surrounded by the tumor, with very little effect was caused by combined therapy with both compounds, muscle tissue remaining especially in the control animals. In resulting in a final tumor volume of 224.03 F 102.31 mm3 some legs, parts of the bone disappeared due to invasive (P < 0.01; 86% inhibition) and a final tumor weight of 376.32 F osteolytic tumor growth. Some samples showed tumor cells in 162.07 mg (P < 0.01; 79% tumor suppression). Tumor volume the lymph vessels (Fig. 3C). doubling time was 7.94 F 2.56 days in controls and was Growth of s.c. Tumors. To compare the efficacy of our extended nonsignificantly by RC-3940-II, MZ-J-7-118, and their analogues for the inhibition of osseous and s.c. tumor growth, we combination to 10.23 F 0.85, 8.72 F 0.75, and 13.20 F 1.28 also implanted PC-3 tumor xenografts s.c. into nude mice days, respectively. bearing intraosseously injected PC-3 cells. At the end of the Binding Assay for BN/GRP and GHRH Receptors. experiment, tumor volumes and tumor weights of s.c. PC-3 Specific receptors for BN/GRP and GHRH were detected on tumors in treated animals were significantly smaller compared membrane fractions of control tumors. In s.c. and orthotopically with controls (Fig. 4). Average tumor volumes and tumor grown PC-3 tumors from the control group, radiolabeled Tyr4- weights were 1,289.62 F 519.36 mm3 and 1,828.66 F 687.76 bombesin was bound to a single class of specific, high-affinity mg in controls. Animals treated with RC-3940-II showed a final binding sites (Kd = 1.33 F 0.11 and 1.15 F 0.13 nmol/L, tumor volume of 427.33 F 120.91 mm3 (P < 0.05) and a final respectively), with a mean maximal binding capacity (maximum

Fig. 2 A, X-rays show tibias of nude mice of control group 6 weeks after PC-3 cell injection into the bone marrow cavity. B, X-rays of legs of nude mice after 4 weeks of treatment with a combination of BN/GRP antagonist RC-3940-II (10 Ag/d) s.c. and GHRH antagonist MZ-J-7-118 (5 Ag/d) s.c. 6 weeks after intraosseous injection of PC-3 cells. C, representative leg of a mouse from the control group 6 weeks after implantation of PC-3 cells. The tumor rapidly replaced the bone marrow cavity and invaded through the bone shaft and into the muscle adjacent to the bone. D, representative leg of an animal that received combined treatment with BN/GRP antagonist RC-3940-II and GHRH antagonist MZ-J-7-118. In most of the animals of this group, the evasion of intraosseously injected PC-3 tumor cells was markedly lower compared with control, and leg circumferences at the end of the experiment were significantly smaller than in controls.

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In vitro Proliferation Studies. To evaluate the direct antiproliferative effect of GHRH antagonist MZ-J-7-118, BN/ GRP antagonist RC-3940-II, and their combination on PC-3 cells in vitro, we used the crystal violet assay. At the concentrations of 1 and 3 Amol/L and following an exposure of 71 hours, MZ-J-7-118 decreased the growth of PC-3 cells by 34% and 38%, respectively (P < 0.001), and after 118 hours by 25% and 80% (P < 0.001), respectively. RC-3940-II at a concentration of 1 and 3 Amol/L and after an exposure of 71 hours reduced in vitro PC-3 cell growth by 11% (P < 0.05) and 17% (P < 0.001), respectively. After 118 hours of exposure, RC-3940-II at 1 and 3 Amol/L decreased PC-3 cell growth by 15% and 25%, respectively (P < 0.001). Treatment with the combination of MZ-J-7-118 and RC-3940-II at equimolar doses of 1 or 3 Amol/L of each antagonist diminished PC-3 cell growth by 23% and 55%, respectively (P < 0.001) after 71 hours of exposure. After an exposure of 118 hours to both compounds at 1 or 3 Amol/L concentration, PC-3 cell growth was decreased by 30% and 78% (P < 0.001), respectively.

DISCUSSION The high mortality and morbidity associated with prostate carcinoma is due mainly to the fact that most patients already have metastatic disease at the time of diagnosis. More than 75% of patients with advanced prostate cancer have bone metastases (4). Because of the limitations of currently available therapeutic modalities for the treatment of metastatic prostate cancer, it is necessary to explore novel options of therapy based on new insights into the mechanisms of metastatic tumor growth. It has been suggested that epidermal growth factor, hepatocyte growth factor/scatter factor, IGF, and osteoblast- derived growth factors may support the proliferation of prostate cancer in the bone (34). Local production of IGF-I and II, epidermal growth factor, vascular endothelial growth factor, basic fibroblast growth factor, and keratinocyte growth factor also aberrantly stimulate the androgen receptor pathway

Fig. 3 Histologic appearance of PC-3 human prostate cancers injected into tibiae of nude mice (H&E staining). A, low-power overview of a control tumor destroying cortical bone and propagating into muscle tissue (120). B, bone fragments surrounded by tumor tissue from the group treated with combination of BN/GRP and GHRH antagonists. There are several osteoclasts (arrows) alongside irregular bone trabecules. In addition, a proliferation of fibroblasts and osteoblasts can be seen (250). C, presence of cancer cells in large numbers in highly dilated lymph vessels (arrowheads) in the muscle tissue of the leg of a control mouse (250).

number of binding sites) of 547.6 F 16.3 and 481.3 F 67.2 fmol/mg membrane protein, respectively. In s.c. and orthotopi- cally grown PC-3 tumors from the control group, radiolabeled GHRH antagonist JV-1-42 was bound to a single class of specific, high-affinity binding sites (Kd = 0.57 F 0.03 and 0.69 F 0.22 nmol/L, respectively), with a mean maximal binding capacity (maximum number of binding sites) of 152.1 F 33.5 Fig. 4 Effect of GHRH antagonist MZ-J-7-118 (5 Ag/d) s.c., BN/GRP and 128.1 F 1 fmol/mg membrane protein, respectively. antagonist RC-3940-II (10 Ag/d) s.c., and their combination on growth of s.c. PC-3 human androgen-independent prostate cancer in nude mice. In competitive displacement experiments on s.c. grown Treatment was started when tumors had grown to 46-53 mm3 and lasted PC-3 tumors, GHRH antagonist MZ-J-7-118 was able to displace for 30 days. Bars, SE. *, P < 0.05, versus control; **, P < 0.01, versus 125 the [ I]JV-1-42 radioligand with an IC50 of 0.15 nmol/L. control.

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(35, 36), contributing to the androgen-independent growth of patients with prostate cancer are osteoblastic rather than prostate cancer cells. Therefore, some strategies for the osteolytic. Thus, PC-3 represents only one aspect of human management of advanced-stage prostate cancer could be based bone metastases and may be inappropriate to represent the on agents that block the production and/or effects of these clinical situation of prostate cancer bone metastasis. However, local growth factors. the PC-3 bone model due to its bone metastatic origin and its Antagonists of BN/GRP and GHRH suppress the growth aggressive growth has been used in previous studies to of human experimental prostate cancers and other malignancies investigate bone metastatic mechanisms and therapeutic agents by mechanisms that include direct antiproliferative (cytostatic) (30, 45). PC-3 cells injected into the tibia rapidly replace bone effects on the cancer cells (16, 23) as well as the inhibition marrow in the bone channel and invade through the bone shaft of various autocrine/paracrine and endocrine growth factors and into the muscle adjacent to the bone (30). Thus, it was (9–11, 21). In our present study, we showed the direct cyto- found that disodiumetidronate, an osteoclast inhibitor em- static effect of BN/GRP and GHRH antagonists on PC-3 cells ployed for the treatment of bone metastasis, failed to reduce cultured in vitro. the incidence, size, or number of bone metastases in a PC-3 Previously, we reported that antagonists of BN/GRP and model (45). GHRH block the synthesis and/or actions of locally produced Because GHRH and BN/GRP antagonists are also being growth factors, such as GHRH, BN/GRP, IGF-I, IGF-II, and tested in other cancers causing osteolytic bone metastases such vascular endothelial growth factor, and down-regulate the as breast and lung cancer, the inhibition of osteolytic lesions in expression of receptors for GRP, epidermal growth factor, and bone may be an important advance in cancer therapy. IGF-I in PC-3, DU-145, and C4-2b androgen-independent as For our initial evaluation of BN/GRP and GHRH well as LNCaP and MDA-PCa-2b androgen-sensitive prostate antagonists in a bone metastatic model, we chose the PC-3 cell cancer models (9–11, 20–23). In the MDA-MB-468 human line, because most of our previous studies with these compounds breast cancer model, GHRH antagonists inhibit the expression of on s.c. tumors in nude mice have been carried out on PC-3. stromal IGF-I, which is a known paracrine growth factor for Compared with other cell lines developed for bone metastatic breast cancer cells (37). Thus, the ability of GHRH antagonists models of prostate cancer such as C4-2 and MDa-PCa-2b that to alter the environment, which surrounds the malignant cells, show osteoblastic bone lesions, PC-3 grows more aggressively, could also contribute to their antiproliferative effect. In prostate and its osteolytic lesions in mouse tibiae can be detected by cancer cells growing orthotopically or intraosseously, the conventional X-rays. Further studies with other cell lines microenvironment surrounding the cells mimics more closely developed for evaluation of prostate cancer, including C4-2 or the situation found in patients than in previously tested s.c. MDa-PCa-2b, will be done soon. models. The ability of the microenvironment to supply the In our study, we found an extensive tumor growth in the hind prostate cancer cells with growth factors could contribute to their limbs into which we injected PC-3 tumor cells. Visible tumors in favorable growth in orthotopic sites and their metastases to the the hind limbs could be observed 3 weeks after the injection. To bone. Some therapeutic agents that were effective in s.c. compare simultaneously the effects of therapy on the s.c. and xenograft models in nude mice worked poorly in humans and intraosseous tumor growth, PC-3 cancers were implanted s.c. and vice versa (38). The lack of predictive value of the s.c. xenograft intraosseously into the same animals. The effect on orthotopic model is due mainly to the essentially nonmetastatic growth tumor growth and metastatic spread was observed in a separate compared with naturally occurring tumors in humans, which experiment. Previously, the s.c. PC-3 model was used for the almost invariably metastasize (38, 39). New experimental assessment of antitumor effects and mechanisms of BN/GRP- models that more accurately reflect the different stages of antagonists and of earlier, less potent GHRH antagonists, such as disease are necessary to speed up the development of new MZ-4-71, MZ-5-156, and JV-1-38 (9, 11, 20, 21, 23). The GHRH therapies for metastatic prostate cancer (39). antagonist employed in the present study, MZ-J-7-118, was Consequently, one of the objectives of this study was to developed recently in our laboratory. MZ-J-7-118 shows extend previous investigations carried out on s.c. xenografts of increased antitumor potency and is used at a dose of 5 Ag/d, PC-3 prostate cancer and to test the effectiveness of BN/GRP whereas earlier antagonists MZ-4-71, MZ-5-156, and JV-1-38 and GHRH antagonists in orthotopic and metastatic models of were given at doses of 20 to 40 Ag/d (9, 11, 20–23). this cell line. The PC-3 cell line was originally isolated from a Our results show that bombesin antagonist RC-3940-II and bone metastasis of a patient with advanced prostate cancer (40). GHRH antagonist MZ-J-7-118 exhibit a strong inhibitory effect It is insensitive to androgens, does not produce prostate-specific at all three tumor sites. The combination therapy with BN/GRP antigen, and grows more aggressively in vivo compared with and GHRH antagonist inhibited orthotopic tumor growth by other frequently used experimental prostate cancer cell lines. 77%, hind limb tumors by 86%, and s.c. tumors by 86%. The Local tumors produced following s.c. injection of PC-3 cells in invasion of the orthotopically implanted tumors to adjacent nude mice seldom metastasize to the bone or other organs (31). organs in the lower abdominal cavity was also powerfully However, when PC-3 cells are inoculated orthotopically, local suppressed by the analogues, especially in the animals receiving tumors are readily produced, which can metastasize to the the combination treatment. The growth of intratibially injected lymph nodes (41–44). The orthotopic, metastatic PC-3 model PC-3 cells, serving as a model for established bone metastases, has been used for testing the antimetastatic effects of various was likewise inhibited. Signs of osteolytic actions like therapeutic agents (42–44). pathologic fractures, or in some cases even complete dissolution In contrast to the osteolytic bone lesions caused by PC-3 of the bone structure, were observed less frequently in treated cells injected into tibiae of nude mice, bone metastases in groups. The present study clearly shows that the BN/GRP and

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Anton Stangelberger, Andrew V. Schally, Jozsef L. Varga, et al.

Clin Cancer Res 2005;11:49-57.

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