The presence of receptors for bombesin/GRP and mRNA for three receptor subtypes in human ovarian epithelial cancers

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Abstract

Bombesin-like peptides can function as autocrine or paracrine growth factors and stimulate the growth of various cancers. The antagonists of bombesin/gastrin-releasing peptide (GRP) suppress the proliferation of diverse tumors including ovarian cancer by mechanisms likely mediated by bombesin receptors. In this study, we used the reverse transcription-polymerase chain reaction (RT-PCR) method to evaluate the mRNA expression of three bombesin receptor subtypes: gastrin-releasing peptide receptor (GRPR), neuromedin B receptor (NMBR), and bombesin receptor subtype 3 (BRS-3), in 22 specimens of human epithelial ovarian cancer and in two human ovarian cancer lines. Of the 22 ovarian cancer specimens analyzed, 17 tumors (∼77%) expressed mRNA for GRPR, 19 (∼86%) showed NMBR mRNA and six (∼27%) revealed BRS-3 mRNA. Thus, 14 of 22 specimens (∼64%) expressed mRNAs for both GRPR and NMBR, and five (∼23%) expressed all three subtypes. The expression of GRPR appeared to be greater in poorly differentiated ovarian carcinomas. A higher incidence of BRS-3 expression was observed in samples with tumor Stage IV (4/4, 100%) compared with Stage III (1/17, ∼6%). mRNA for both GRPR and NMBR was also detected in OV-1063 and UCI-107 human ovarian cancer xenografts, but BRS-3 was found only in OV-1063, which originated from a metastatic tumor. In addition, functional receptors for bombesin/GRP were found in eight of 11 ovarian cancer specimens investigated and in both ovarian cancer lines by receptor binding assay. Our study indicates that GRPR and NMBR are widely distributed in human ovarian carcinomas with BRS-3 being found in Stage IV tumors. Some approaches based on bombesin/GRP receptor antagonists or targeted bombesin analogs could be considered for treatment of ovarian cancers.

Introduction

Bombesin (Bn)-like peptides have been shown to stimulate the growth of various human cancers, such as small cell lung carcinoma (SCLC), and pancreatic, breast, prostatic, and gastric cancer [1], [2], [3], [4], [5], [6]. These effects are mediated by the receptors for bombesin, which are G-protein coupled. Molecular cloning revealed the presence of three distinct bombesin receptor subtypes in humans and rodents: the gastrin-releasing peptide receptor (GRPR), the neuromedin B receptor (NMBR) and the bombesin receptor subtype 3 (BRS-3) [7], [8], [9]. The gastrin-releasing peptide and neuromedin B bind to GRPR and NMBR, respectively, with high affinity, while BRS-3 responds only to relatively high concentrations of known Bn-like peptides [7], [8], its natural ligand being still unknown. These receptors share about 50% amino acid sequence identity and are involved in growth regulation [7], [9].

Several therapeutic approaches based on hormonal peptide analogs have been proposed for the treatment of various cancers [10] and numerous bombesin/GRP receptor antagonists have been developed [11], [12], [13]. Bombesin/GRP antagonists RC-3095 and RC-3940-II, synthesized in our laboratory [11], inhibit growth of various human cancers including breast, gastric, prostatic, colorectal, SCLC, and pancreatic cancer xenografted into nude mice [4], [6], [14], [15], [16], [17].

Ovarian cancer is the fifth most common cancer among women in the United States. Five-year survival rates are very poor (∼21%) for late-stage disease (stages III and IV) [18]. The current treatment of ovarian cancer is mainly based on debulking surgery combined with chemotherapy, but the long-term outcome of such therapies is disappointing [18], [19], [20]. Recently, we found that bombesin/GRP antagonists RC-3095 and RC-3940-II can inhibit the growth of OV-1063 human ovarian cancer cell line xenografted into nude mice (Chatzistamou and Schally, in preparation), indicating that these compounds could be considered as potential agents for treatment of ovarian cancer.

The antitumor effects of some hormonal peptide analogs depend on the presence of their specific receptors on tumor cells [10]. The identification of the expression pattern for Bn-like peptide receptors in primary tumors is essential for the prediction of the efficacy of treatment based on bombesin/GRP antagonists. In this study, we evaluated the mRNA expression of GRPR, NMBR and BRS-3 subtypes by reverse transcription-polymerase chain reaction (RT-PCR) in 22 human ovarian tumor specimens and two human ovarian cancer cell lines xenografted into nude mice.

Section snippets

Cancer cell lines and implantation of tumors into nude mice

Human ovarian cancer cell line OV-1063, human SCLC cell lines H345 and H69 were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The UCI-107 human epithelial ovarian cancer cell line was provided by Dr. A. Manetta (University of California, Irvine Medical Center, Orange, CA, USA). These cancer cells were routinely maintained in 250-ml flasks in RPMI-1640 medium containing 10% fetal bovine serum, 1 mM pyruvate, 1:100 minimal essential medium vitamins, 2 mM

Results

When RT-PCR with specific primers (Table 2) was used to evaluate the mRNA expression of GRPR, NMBR and BRS-3 genes in 22 human ovarian cancer specimens, it produced fragments of expected size of 158 bp for GRPR, 484 bp for NMBR and 375 bp for BRS-3, respectively (Fig. 1). The respective PCR products could be detected in 17 of 22 (∼77%) ovarian carcinomas for GRPR, 19 of 22 (∼86%) for NMBR, and six of 22 (∼27%) for BRS-3 (Table 1). These results demonstrate that GRPR and NMBR are the most

Discussion

We investigated the mRNA expression of the three bombesin receptor subtypes in human ovarian carcinomas by RT-PCR, a highly sensitive and specific technique for the amplification of small quantities of mRNA. Primer pairs for the PCR amplification of each subtype were chosen to span an intron to exclude genomic DNA contamination. Because of the low expression of GRPR and BRS-3 in most samples, nested PCR was applied for identifying these two genes.

Our results showed that mRNAs for GRPR (17/22,

Acknowledgements

We thank Dr. William R. Robinson (Tulane Cancer Center) for supplying human ovarian cancer specimens. We are grateful to Drs. Zsuzsanna Kahán for collection of tumors, Valér J. Csernus and Rhonda D. Kineman for clinical support, and Hippokratis Kiaris and Artur Plonowski for help in preparation of the manuscript. The work described in this paper was supported by the Medical Research Service of the Veterans Affairs Department and a grant from ASTA Medica (Frankfurt on Main, Germany) to Tulane

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