Inhibition of growth of ES-2 human ovarian cancers by bombesin antagonist RC-3095, and luteinizing hormone-releasing hormone antagonist Cetrorelix

Cancer Letters 171 (2001) 37±45

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Inhibition of growth of ES-2 human ovarian cancers by bombesin antagonist RC-3095, and luteinizing hormone-releasing hormone antagonist Cetrorelix Ioulia Chatzistamou a,b, Andrew V. Schally a,b,*, Karoly Szepeshazi a,b, Kate Groot a, Francine Hebert a, Jose M. Arencibia a,b a

Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center, 1601 Perdido Street, New Orleans, LA 70112, USA Section of Experimental Medicine, Department of Medicine, Tulane University School of Medicine, New Orleans, LA 70112, USA

b

Received 30 November 2000; received in revised form 30 March 2001; accepted 2 April 2001

Abstract We evaluated the effects of the bombesin/gastrin-releasing peptide (GRP) antagonist RC-3095, and the luteinizing hormone± releasing hormone (LH±RH) antagonist Cetrorelix, administered singly or in combination, on the growth of human ovarian carcinoma cell line ES-2, xenografted into nude mice. RC-3095 at a dose of 20 mg/day and Cetrorelix (100 mg/day), signi®cantly reduced the volume of ES-2 tumors by 63.0% (P , 0:01) and 38.0% (P , 0:05) respectively, after 44 days of treatment, as compared with controls. The combination of RC-3095 with Cetrorelix inhibited the growth of ES-2 tumors by 66.2% (P , 0:01). Serum levels of LH were signi®cantly decreased in the groups treated with Cetrorelix alone and/or in combination with RC-3095. RT-PCR analyses revealed that the expression of mRNA for receptors of GRP (GRPR/BRS-1) and Neuromedin B (NMBR/BRS-2) on tumors was signi®cantly decreased in all the treated groups. The expression of mRNA for epidermal growth factor receptors (EGFR) on tumors was reduced by 36.5 % (P , 0:05) in the animals treated with Cetrorelix and by 72.5% (P , 0:05) in the group that received the combination of RC-3095 with Cetrorelix. Our results indicate that the bombesin antagonist RC-3095 and the LH-RH antagonist Cetrorelix inhibit effectively the growth of ES-2 ovarian cancers in nude mice. These antagonists and their combination could be considered for the therapy of patients with ovarian cancer. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cancer therapy; Ovarian tumors; Bombesin/gastrin-releasing peptide; Luteinizing hormone±releasing hormone

1. Introduction Epithelial ovarian carcinoma is the most frequent cause of death from gynecological cancers among women in the Western World [1]. Treatment of

* Corresponding author. Tel.: 11-504-589-5230; fax: 11-504566-1625.

patients with advanced ovarian cancer is based on debulking surgery followed by aggressive chemotherapy or radiation. However, the results are not satisfactory and new therapeutic strategies should be explored. Bombesin-like peptides, such as the gastrin releasing peptide (GRP), were initially reported as autocrine growth factors in the development and progression of human small cell lung carcinomas [2±4], but recent studies also demonstrate an invol-

0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00543-2

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I. Chatzistamou et al. / Cancer Letters 171 (2001) 37±45

vement of bombesin and GRP in the pathogenesis of pancreatic, prostatic, and breast cancers and glioblastomas [5±9]. Speci®c receptors for bombesin/GRP have been demonstrated in various human cancers including ovarian cancer specimens and ovarian cancer cell lines [10±12]. So far, three subtypes of bombesin/GRP receptors have been characterized in humans [13]. Bombesin receptor subtype-1 (GRPR/ BRS-1) binds GRP with high af®nity, while NMBR/ BRS-2 is preferentially activated by neuromedin B [13]. BRS-3 is considered an orphan receptor with unknown natural ligand. Recently, a fourth receptor subtype BRS-4 has been cloned and characterized in amphibia, but its existence in human remains to be demonstrated [14]. The ®ndings that bombesin-like peptides may function as autocrine/paracrine growth factors in certain tumors stimulated the development of bombesin/GRP antagonists as potential antitumor agents [12,15]. Bombesin/GRP antagonists synthesized in our laboratory suppress the growth of various experimental tumors including prostatic, breast, ovarian, lung, pancreatic, and gastric cancers, and glioblastomas [9,16±25]. It is also well established that receptors for luteinizing hormone±releasing hormone (LH±RH) are expressed by ovarian cancers [26±29]. Chronic treatment with the LH-RH antagonist Cetrorelix [26] produces a signi®cant inhibition of the OV-1063 human epithelial ovarian cancer, xenografted into nude mice. This inhibition of tumor growth could be due to the suppression of the pituitary-gonadal axis, but Cetrorelix may also suppress tumors by direct mechanisms that involve the blockade of the autocrine/paracrine effects of LH-RH on the cancer cells. Thus, LH-RH could affect growth of ES-2 human ovarian cancer cell line, since both LH-RH and LH-RH receptors appear to be expressed in this cell line and the growth of ES-2 cells cultured in vitro is suppressed by Cetrorelix [30]. In the present study we evaluated the antitumor effects of bombesin/GRP antagonist RC-3095 and LH-RH antagonist Cetrorelix on the growth of human ovarian cancer cell line ES-2 xenografted into nude mice. The effects of the treatment on the levels of mRNA for bombesin/GRP receptors, as well as EGF and LH-RH receptors on tumors were also evaluated.

2. Materials and methods 2.1. Peptides and reagents Bombesin antagonist d-Tpi 6,Leu 13C[CH2NH]Leu 14BN(6-14), (RC-3095) originally synthesized in our laboratory [31], was manufactured by ASTA Medica (Frankfurt am Main, Germany). RC-3095 acetate (D22213) was used for treating nude mice. Cetrorelix (SB-75), [Ac-d-Nal(2) 1, d-Phe(4Cl) 2, dPal(3) 3, d-Cit 6, d-Ala 10]LH-RH, also originally synthesized in this laboratory [32] was obtained from ASTA Medica (Frankfurt am Main Germany). For daily injections, RC-3095 was dissolved in dimethylsulfoxide (DMSO) and diluted with 0.9% saline. The ®nal concentration of DMSO was 0.1%. Cetrorelix was dissolved in distilled water containing 5% mannitol. 2.2. Animals Six-week-old female athymic NCR/c (nu/nu) nude mice were purchased from Frederick Cancer Research Facility of the National Cancer Institute (Frederick, MD) and housed in a laminar air¯ow cabinet under pathogen-free conditions with a 12-h light/12-h dark schedule and fed autoclaved standard chow and water ad libitum. Their care was in accord with institutional guidelines. 2.3. Cell line Human ovarian cancer cell line ES-2, a poorly differentiated ovarian clear cell carcinoma derived from a tumor of a 47-year-old African±American female [33], was obtained from American Type Culture Collection (Manassas, VA). It was maintained in McCoy 5A medium, supplemented with 10% fetal bovine serum (FBS), vitamins, antibiotics, and antimycotics as described previously [22]. Cells were cultured in Costar T-75 ¯asks in a humidi®ed atmosphere of 5% CO2 and 95% air at 378C and passed every 4±6 days using 0.25% trypsin-EDTA [22]. 2.4. Experimental protocol Xenografts of ES-2 cells were initiated by s.c. injection of 10 7 cells into the right ¯anks of three female nude mice. ES-2 tumors resulting after four

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weeks were aseptically dissected and mechanically minced; 1 mm 3 tumor pieces were transplanted subcutaneously by trocar needle into the right ¯anks of nude mice. Two weeks after transplantation, when tumors reached a volume of approximately 20±30 mm 3, the mice were randomly divided into ®ve experimental groups of 6±8 animals each and received the following treatment as daily s.c. injections: group 1 (control), vehicle solution; group 2, bombesin/GRP antagonist RC-3095 at a dose of 20 mg/day per animal; group 3, LH-RH antagonist Cetrorelix, 100 mg/day per animal; group 4, the combination of RC3095 at 20 mg/day and Cetrorelix at 100 mg/day per animal. The treatment was continued for 44 days. Tumors were measured once a week with microcalipers and the tumor volume was calculated as length £ width £ height £ 0.5236 [34]. At the end of the experiment, mice were anesthetized with methoxy¯urane (Metofane; Pitman-Moore, Mundelein, IL) and sacri®ced by decapitation, and trunk blood was collected. The serum was separated and analyzed by radioimmunoassay. Body weights were recorded and various organs were removed and weighed. Tumors were dissected, cleaned, and weighed and samples were stored in 2708C for molecular biology analyses. 2.5. Histological methods Samples of tumor tissue were ®xed in 10% buffered formalin. Specimens were embedded in Paraplast (Oxford Labware, St. Louis, MO). Six micron-thick sections were cut and stained with hematoxylin± eosin. Mitotic and apoptotic cells were counted in 10 standard high power microscopic ®elds containing on the average 250 cells, and their numbers per 1000 cells were accepted as the mitotic and apoptotic indices, respectively. The area of necrosis was estimated in histological slides made at the largest cross section of the tumors by the point-counting method. For the demonstration of the nucleolar organizer regions (NOR) in tumor cell nuclei, the AgNOR method was used as described previously [35]. The silver-stained black dots in 50 cells of each tumor were counted and the AgNOR number per cell was calculated. The data were evaluated by one way analysis of variance and Dunnett's method using a SigmaStat computer program.

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2.6. Radioimmunoassays for LH and EGF Serum LH was determined by radioimmunoassay (RIA) using materials provided by the National Hormone & Pituitary Program, (NHPP, Rockville, MD) (mouse LH-RP/AFP-5306A/, rat LH-I-9/AFP10250C/, anti-rat LH-RIA-11/AFP C 697071P). Interassay and intra-assay coef®cients of variation were less than 15 and 10%, respectively, for the assay. EGF was extracted from serum by a modi®ed acid± ethanol extraction and measured as described [18]. 2.7. RNA Extraction and Reverse-Transcription-PCR (RT-PCR) Total RNA was extracted from human ES-2 tumors by using RNAzol B (Tel-Test Inc., Friendwood, TX) according to the manufacturer's instructions. RNA was reverse transcribed into cDNA and subsequently subjected to semiquantitative PCR for the evaluation of the mRNA expression for b-actin (internal control), GRPR, NMBR, BRS-3, EGFR (EGF receptors) and LH-RH receptors as described previously [22,29,36]. The oligonucleotide primers used and the size of the RT-PCR products are described in Table 1 [22,29]. Aliquots of PCR-ampli®ed product was resolved by electrophoresis on a 1.8% agarose gel, stained with ethidium bromide, and visualized under ultraviolet light. Quanti®cation of PCR-ampli®ed product was accomplished using a scanning densitometer (model GS-700, Bio-Rad) coupled with the Bio-Rad personal computer analysis software. 2.8. Statistical analyses Data are expressed as mean ^ SE. Statistical analyses were performed using Duncan's new multiple range test and Student's two-tailed t-test, unless otherwise speci®ed. All P-values are based on twosided hypothesis testing [37]. 3. Results 3.1. Effects of treatment with bombesin/GRP antagonist RC-3095 and LH-RH antagonist Cetrorelix on growth of ES-2 tumors in nude mice After 44 days of treatment, the volume of ES-2

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I. Chatzistamou et al. / Cancer Letters 171 (2001) 37±45

Table 1 Nucleotide sequence and size of the expected PCR products for oligonucleotide primers used for RT-PCR on receptors for gastrin releasing peptide (GRP), bombesin subtype 3 (BRS-3), neuromedin B (NMBR), LH-RH and EGF Gene

Sequence

PCR product (bp)

0

b-actin

0

5 -TCATGAAGTGTGACGTGGAC-3 5 0 -ACCGACTGCTGTCACCTTCA-3 0 5 0 -ATCTTCTGTACAGTCAAGTCC-3 0 5 0 -GCTTTCTCATGGAAGGGATG-3 0 5 0 -ATTTGGCAGGATTGGCTGC-3 0 5 0 -TGAGGCAGATCTTCATCAG-3 0 5 0 -GGCTCAAAGGCAGCCTCACT-3 0 5 0 -AGTCTTCAGGATGGCATTGG-3 0 5 0 -GCTCTGTGGTTTCTAACG-3 0 5 0 -CTGCCTTGTATCTGTCAGC-3 0 5 0 -CGGACTCTGCTGGAAAGGA-3 0 5 0 -GACGTCTGCATGTCCATGG-3 0 5 0 -GACCTTGTCTGGAAAGATCC-3 0 5 0 -CAGGCTGATCACCACCATCA-3 0 5 0 -ACGCAGATAGTCGCCCAAAGTTCC-3 0 5 0 -AGGAAGGTGTCGTCTATGCTGTCC-3 0

GRPR Outer GRPR Internal BRS-3 Outer BRS-3 Internal NMBR LH-RH-R EGFR

tumors in the groups receiving the bombesin/GRP antagonist RC-3095 alone was signi®cantly reduced to 775.2 ^ 105.0 mm 3 (P , 0:01) and by combination of RC-3095 with Cetrorelix to 707.6 ^ 124.6 mm 3 (P , 0:01), indicating decreases of 63.0 and 66.2% in tumor volume, respectively (Table 2, Fig. 1) as compared with the controls which measured 2094.1 ^ 251.3 mm 3. In the group treated with LHRH antagonist Cetrorelix alone, the tumor volume was reduced to 1298.0 ^ 48.2 mm 3, corresponding to a 38.0% (P , 0:05) decrease (Table 2, Fig. 1). The ®nal tumor weights were also signi®cantly diminished in the group injected with RC-3095 or its combination with Cetrorelix by 45.6% (P , 0:05) and 50.0% (P , 0:05) respectively, and in the group receiving Cetrorelix alone by 27.4% (P , 0:05), as

459 375 158 489 375 484 319 400

compared to controls (Table 2). Signi®cant (P , 0:05) decreases in the weight of ovaries were observed in the groups that received Cetrorelix alone or in combination with RC-3095 (data not shown). No signi®cant differences in body weights or in the weights of various organs, such as liver, kidneys, and heart, were observed between the control and the treated animals. 3.2. Histological ®ndings ES-2 human ovarian cancers were undifferentiated tumors consisting of relatively large cells without any special arrangement. The cells had large nuclei showing considerable polymorphism. The amount of stroma was minimal. The results of the histological analysis

Table 2 Effect of treatment with bombesin/GRP antagonist RC-3095 and LH-RH antagonist Cetrorelix on tumor volume and weight in nude mice bearing xenografts of ES-2 human ovarian carcinoma cell line Treatment

Initial tumor volume (mm 3)

Final tumor volume (mm 3)

Tumor weight (g)

Tumor burden (mg/g b.w.)

Control RC-3095 Cetrorelix RC-3095 1 Cetrorelix

22.4 ^ 3.3 21.8 ^ 6.0 21.0 ^ 1.9 20.7 ^ 3.3

2094.1 ^ 251.3 775.2 ^ 105.0 a 1298.0 ^ 48.2 b 707.6 ^ 124.6 a

2.30 ^ 0.23 1.25 ^ 0.21 b 1.67 ^ 0.04 b 1.15 ^ 0.26 b

83.7 ^ 9.2 48.8 ^ 9.0 b 60.3 ^ 1.7 b 42.4 ^ 10.2 b

a b

P , 0:01 vs. controls. P , 0:05 vs. controls.

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3.3. Effects of treatment with bombesin/GRP antagonist RC-3095 and LH-RH antagonist Cetrorelix on serum levels of LH and EGF in nude mice bearing ES-2 tumors Serum LH and EGF levels in controls and in animals treated with RC-3095 and Cetrorelix alone and in combination were measured (Table 4). There were no signi®cant changes in EGF serum levels after chronic treatment with RC-3095 and Cetrorelix alone or in combination compared to controls. Serum levels of LH were decreased to 0.32 ^ 0.03 ng/ml (P , 0:004) and 0.28 ^ 0.06 ng/ml (P , 0:002) in groups treated with Cetrorelix alone or in combination with RC-3095 as compared to 1.39 ^ 0.23 ng/ml for the controls.

Fig. 1. Tumor volumes in athymic female nude mice bearing s.c. transplanted ES-2 human epithelial ovarian cancer cell line during treatment with bombesin/GRP antagonist RC-3095 at a dose of 20 mg/animal and LH-RH antagonist Cetrorelix at a dose of 100 mg/ animal administered by daily s.c. injections. Vertical bars represent SE **P , 0:01, *P , 0:05 vs. control.

are summarized in Table 3. Bombesin/GRP antagonist RC-3095 and LH-RH antagonist Cetrorelix alone or in combination decreased signi®cantly (P , 0:05) the AgNOR numbers in the ES-2 tumors as compared with the controls. The combination of RC-3095/ Cetrorelix signi®cantly increased (P , 0:05) the apoptotic index while mitotic index was signi®cantly decreased (P , 0:05) only in the RC-3095 group. The ratio of apoptotic to mitotic indices in ES-2 tumors was increased in the RC-3095/Cetrorelix groups, compared to controls.

3.4. Investigation of the expression of mRNA for bombesin/GRP, EGF and LH-RH receptors in ES-2 human ovarian cell carcinoma The expression of mRNA for bombesin/GRP receptor subtypes BRS-1 (GRPR), BRS-2 (NMBR), BRS3, as well as for epidermal growth factor receptor (EGFR) and LH-RH receptor in ES-2 tumors was evaluated by RT-PCR. As shown in Fig. 2, in the RC-3095 and the Cetrorelix injected groups, mRNA for GRPR was signi®cantly reduced by 26.6% (P , 0:02) and by 49.7% (P , 0:0004) respectively, while in the RC-3095/Cetrorelix treated group, the GRPR mRNA expression was suppressed by 86.7% (P , 0:0001). mRNA for NMBR was also signi®cantly decreased in all the treated groups (Fig. 2) the reduction being 46.6% (P , 0:02) in the group receiving RC-3095, 38.9% (P , 0:03) in the Cetrorelix treated group, and 84.8% (P , 0:001) in the

Table 3 Effect of treatment with bombesin/GRP antagonist RC-3095 and LH-RH antagonist Cetrorelix on various histological parameters of ES-2 human ovarian carcinoma cell line xenografted into nude mice Groups

% Area of necrosis

Mitotic index

Apoptotic index

Ratio of apoptotic to mitotic indices

No. of AgNORs per cell

Control RC-3095 Cetrorelix RC-3095 1 Cetrorelix

54 ^ 10 58 ^ 9 61 ^ 8 70 ^ 5

14.7 ^ 1.8 6.5 ^ 0.6 a 11.8 ^ 1.9 10.2 ^ 1.3

8.1 ^ 0.6 8.5 ^ 1.8 11.7 ^ 1.4 15.4 ^ 1.6 a

0.61 ^ 0.09 1.35 ^ 0.33 1.05 ^ 0.13 1.63 ^ 0.35 a

7.46 ^ 0.08 6.60 ^ 0.04 a 6.76 ^ 0.12 a 6.20 ^ 0.13 a,b

a b

P , 0:05 vs. control. P , 0:05 vs. Cetrorelix treated group.

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Table 4 Effect of treatment with bombesin/GRP antagonist RC-3095 and LH-RH antagonist Cetrorelix on serum LH and EFG levels in nude mice bearing xenografts of ES-2 human ovarian carcinoma cell line Treatment

LH (ng/ml)

EGF (ng/ml)

Control RC-3095 Cetrorelix RC-3095 1 Cetrorelix

1.39 ^ 0.23 0.67 ^ 0.04 0.32 ^ 0.03 a 0.28 ^ 0.06 a

3.45 ^ 0.50 5.06 ^ 0.63 4.66 ^ 1.25 5.24 ^ 0.73

a

P , 0:001 vs. controls.

action of both these antagonists is in agreement with the decrease in the expression of AgNOR numbers in ES-2 tumors. AgNOR is a marker of cell proliferation and its expression is increased in highly proliferative tissues. In agreement with our earlier reports on pancreatic, gastric, mammary, prostatic, and lung cancers, the inhibition of tumor growth by bombesin/GRP antagonists was accompanied by a marked down-regulation of receptors for bombesin/GRP and EGF, as re¯ected by major fall in mRNA levels for these receptors [16± 19,23±25]. In our study, however, RC-3095 used

group injected with the combination of RC-3095/ Cetrorelix. Only a few of the ES-2 tumors tested appeared to express BRS-3 and there was no difference in the mRNA levels for BRS-3 as shown in Fig. 2. The expression of mRNA for EGFR and LH-RH receptors were also investigated by RT-PCR analysis. RC-3095 alone had no signi®cant effect, but the expression of EGFR mRNA was signi®cantly reduced by 36.5 % (P , 0:05) in the Cetrorelix treated group and by 72.3 % (P , 0:05) in the RC-3095/Cetrorelix combination group (Fig. 2). There was a slight decrease (not signi®cant) in the expression levels of mRNA for LH-RH receptors in the groups treated with Cetrorelix when compared to the control group, while no effect was observed after treatment with RC3095 alone (Fig. 2). 4. Discussion The present methods for treatment of patients with epithelial ovarian cancer are not satisfactory and new therapeutic modalities must be developed. In view of ®ndings that bombesin/GRP receptors and LH-RH receptors are expressed by ovarian cancers [10,11,26,29,30], we carried out the current study to compare the inhibitory effect of bombesin/GRP antagonist RC-3095 and LH-RH antagonist Cetrorelix on the growth of ES-2 human ovarian cancers xenografted into athymic nude mice. Both of these antagonists strongly inhibited the growth of ES-2 tumors in nude mice producing a signi®cant reduction in the ®nal tumor volume and tumor weight. RC-3095 proved to be more powerful than Cetrorelix in suppressing growth of ES-2 tumors. The antitumor

Fig. 2. Expression of Bombesin receptors subtypes, EGFR and LHRHR in ES-2 human ovarian cancers grown in nude mice. mRNA was reverse transcribed and ampli®ed by PCR. The products were electrophoresed in 1.8% agarose gels and stained with ethidium bromide. The expression of b-actin was used as internal control. M: 100 bp molecular size marker. (a) control group; (b) RC-3095 treated group; (c) cetrorelix treated group; (d) combination of RC3095 and Cetrorelix treated group. The size of the expected products is shown in parentheses.

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alone had no effect on mRNA expression for EGFR. Nevertheless, when RC-3095 was used in combination with Cetrorelix, the decrease in EGFR expression was much greater than that produced by Cetrorelix alone. We have recently reported the inhibition of tumor growth by bombesin analogs RC-3095 and RC-3940-II in xenografts of human breast cancer MDA-MB-468, which overexpresses EGFR, but the analyses by RT-PCR revealed no decrease in EGFR mRNA expression [38]. Because Cetrorelix alone causes a decrease of 36.5% in EGFR mRNA expression, the greater inhibition achieved after treatment of RC-3095 in combination with Cetrorelix, could be due to the fact that RC-3095 can only affect EGFR mRNA expression after Cetrorelix has initially downregulated a possible over-expression of this receptor. The inhibition of growth of ES-2 tumors by Cetrorelix is most probably due to both direct and indirect mechanisms. The indirect mechanism involves the suppression of the pituitary-gonadal axis, as demonstrated by the signi®cant reduction in the serum LH levels in the Cetrorelix treated animals, while the direct mechanism may involve the blockade of the autocrine/paracrine production of LH-RH by the cancer cells. This would be in agreement with previous ®ndings on the suppression of growth of OV-1063 tumors by Cetrorelix, but not by the LHRH agonist triptorelin. [22,26]. As both types of LH-RH analogues induced a comparable suppression of the pituitary-gonadal axis, it was suggested that the antitumor effect of Cetrorelix was exerted directly on LH-RH receptors in tumors [26]. Recently we showed that LH-RH is produced and secreted by the ES-2 cells cultured in vitro and that Cetrorelix inhibited their proliferation, suggesting that LH-RH may function as a growth factor for ES-2 ovarian cancer cell line [30]. Since bombesin/GRP antagonist RC-3095 and LHRH antagonist Cetrorelix inhibit tumor growth by different mechanisms, it would be expected for the combination of these antitumor agents to produce a greater antitumor effect than when these agents are administered singly. Although in the present experiments the combination of RC-3095/Cetrorelix produced a somewhat stronger inhibition of tumor growth than RC-3095 alone, no signi®cant and striking difference was found. This may be due to the fact that a nearly maximal inhibition of tumor growth was

43

produced by RC-3095 alone and this suppression could not be further increased by a combination with Cetrorelix. If this is the reason for the absence of synergism between these compounds, we would expect that the repetition of these experiments with lower doses could unmask this expected synergistic effect between Cetrorelix and RC-3095 in inhibiting tumor growth. In summary, our results show that LH-RH antagonist Cetrorelix and bombesin/GRP antagonist RC3095 strongly inhibit the growth of ES-2 ovarian cancers. These agents could be considered for the development of novel therapeutic modalities for patients with some types of ovarian carcinoma. Acknowledgements We thank Elena Glotser and Anita Feil for technical assistance. We also thank Dr A.F. Parlow (NIDDK'S National Hormone & Pituitary Program) for providing the materials used in RIA. The work described in this paper was supported by the Medical Research Service of the Veterans Affairs department (to A.V.S.) and by a grant from ASTA Medica (Frankfurt am Main, Germany) to Tulane University School of Medicine (to A.V.S.). References [1] S.H. Landis, T. Murray, S. Bolden, P. Wingo, Cancer Statistics, CA Cancer J. Clin. 49 (1999) 8±31. [2] D.N. Carney, F. Cuttitta, T.W. Moody, J.D. Minna, Selective stimulation of small cell lung cancer, Nature 316 (1987) 823± 825. [3] T.W. Moody, F. Cuttitta, Growth factor and peptide receptors in small cell lung cancer, Life Sci. 52 (1993) 1161±1173. [4] J.M. Siegfried, Y. Han, M.A.A. DeMichele, J.D. Hunt, A.L. Gaither, F. Cuttitta, Production of gastrin-releasing peptide by a non-small cell lung carcinoma cell line adapted to serumfree and growth factor-free conditions, J. Biol. Chem. 269 (1994) 8596±8603. [5] M.C. Bologna, C. Festuccia, P. Muzi, L. Biordi, M. Ciomei, Bombesin stimulates growth of human prostatic cancer cells in vitro, Cancer 63 (1989) 1714±1720. [6] T. Yano, J. Pinski, K. Groot, A.V. Schally, Stimulation by bombesin and inhibition by bombesin/gastrin releasing peptide antagonist RC-3950 of growth of human breast cancer cell lines, Cancer Res. 54 (1992) 4545±4547. [7] Q.J. Wang, A.J. Knezetic, A.V. Schally, P.M. Pour, T. Adrian, Bombesin may stimulate proliferation of human pancreatic

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