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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-2179
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 6 3614-3621
Copyright © 2005 by The Endocrine Society

Inhibition of Growth of Experimental Human Endometrial Cancer by an Antagonist of Growth Hormone-Releasing Hormone

Jörg B. Engel, Gunhild Keller, Andrew V. Schally, Gabor L. Toller, Kate Groot, Alexandre Havt, Patricia Armatis, Marta Zarandi, Jozsef L. Varga and Gabor Halmos

Endocrine, Polypeptide, and Cancer Institute (J.B.E., G.K., A.V.S., G.L.T., K.G., A.H., P.A., M.Z., J.L.V., G.H.), Veterans Affairs Medical Center; and Section of Experimental Medicine (J.B.E., G.K., A.V.S., G.L.T., K.G., A.H., P.A., M.Z., J.L.V., G.H.), Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112

Address all correspondence and requests for reprints to: Dr. Andrew V. Schally, Veterans Affairs Medical Center, 1601 Perdido Street, New Orleans, Louisiana 70112-1262. E-mail: aschally{at}tulane.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Antagonists of GHRH are being developed for the treatment of various cancers. In this study we investigated in vivo and in vitro the effects of the GHRH antagonist MZ-J-7-118 and its mechanism of action in HEC-1A human endometrial cancer. Treatment of nude mice bearing HEC-1A xenografts with 10 µg/d MZ-J-7-118 for 6 wk significantly inhibited the volume of HEC-1A tumors by 43%, tumor weight by 40% compared with controls and prolonged the tumor doubling time from 18.7 ± 1.4 to 25.4 ± 3.8 d. Administration of 20 µg MZ-J-7-118, sc, twice a day significantly (P < 0.05) decreased HEC-1A growth, as evidenced by a 57.9% decrease in tumor volume, a 50.7% reduction in tumor weight, and the extension of tumor doubling time from 17.5 ± 2.8 to 36.4 ± 6.5 d. Therapy with GHRH antagonists significantly decreased serum IGF-I levels in experiment 1, and significantly increased tumoral IGF-I levels in experiment 2 in treated mice. Levels of IGF-II and vascular endothelial growth factor-A in tumors were not changed. Specific high affinity binding sites for GHRH were found on HEC-1A tumor membranes using ligand competition assays with 125I-labeled GHRH antagonist JV-1-42. MZ-J-7-118 displaced radiolabeled JV-1-42 with an IC50 of 0.13 ± 0.04 nM. The expression of mRNA for GHRH and splice variants of the GHRH receptor in HEC-1A tumors was demonstrated by real-time RT-PCR analysis. HEC-1A cells cultured in vitro secreted GHRH into the medium. The GHRH antagonist MZ-J-7-118 inhibited the growth of HEC-1A cells in vitro. Our results indicate that GHRH antagonists can reduce the growth of human endometrial cancer and could be used as an alternative adjuvant therapy for the management of endometrial cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOMETRIAL CARCINOMA IS the most common malignancy of the female genital tract, and it ranks fourth in incidence among all cancers in American women (1, 2, 3, 4, 5). Approximately 40,000 new cases of endometrial cancer are diagnosed annually in the United States, with about 7,000 deaths occurring from this disease (1, 2, 3, 4, 5). Thus, endometrial carcinoma is the eighth leading cause of mortality from cancer in women (1, 2, 3, 4, 5).

In nearly 75% of the cases, the tumor is confined to the uterine corpus, and primary surgery, followed by radiation, is the conventional treatment strategy. In the case of advanced or recurrent disease, systemic hormonal therapy with progestagens or combination chemotherapy are used with substantial response rates (1, 2, 4, 5).

When diagnosed at early stages, endometrial cancer has an excellent prognosis, with 5-yr survival rates of 87% and 76% for International Federation of Gynecology and Obstetrics (FIGO) stages I and II, respectively (4). However, 5-yr survival rates decrease to 59% in FIGO stage III and 18% in FIGO stage IV disease (4). The overall survival in patients with recurrent endometrial cancer is very low (7.7%), and the prognosis is dismal (6). Therefore, novel effective therapeutic approaches are required for the treatment of disseminated and relapsed endometrial carcinoma.

A large number of antagonistic analogs of GHRH were synthesized in our laboratory in recent years in an endeavor to develop a new class of antineoplastic agents (7, 8, 9, 10). It has been determined that GHRH antagonists inhibit the growth of various experimental human cancers, such as pancreatic (11), colorectal (12), prostatic (13, 14, 15, 16), breast (15), and renal (17) cancers; glioblastomas (18); osteosarcomas and Ewing sarcomas (19, 20); small cell lung carcinomas; and non-small cell lung carcinoma (21, 22, 23), but their effects on endometrial cancers have not been studied.

GHRH antagonists suppress tumor growth through indirect and direct pathways. The indirect, endocrine mechanism operates through the suppression of GH release from the pituitary, and the resulting inhibition of hepatic production of IGF-I (7, 8). An inhibition of prostatic, renal, and lung cancers and bone tumors by high doses of the GHRH antagonists MZ-4-71 and MZ-5-156 was associated with reduced hepatic and serum IGF-I levels (13, 14, 17, 19, 23). IGF-I is an established mitogen for various cancers (24); thus, the decrease in the level of IGF-I in serum is likely to contribute to the inhibition of tumor growth. However, in other in vivo studies using lower doses of GHRH antagonists or more recently developed analogs with different structural features, such as antagonists JV-1-36, JV-1-38 and MZ-J-7-118, the growth of human pancreatic, colorectal, prostatic, breast, ovarian, and lung cancers was inhibited in the absence of any significant effects on serum IGF-I, (11, 12, 21, 22, 25, 26, 27). These studies led to the conclusion that the main mechanism responsible for tumor inhibition could be a direct action of the antagonists on the tumor tissue (7, 10). In addition, it was observed that GHRH antagonists can inhibit the proliferation of diverse cancer lines by direct action in vitro under conditions in which the contribution of the hypothalamic GHRH/pituitary GH/hepatic IGF-I axis is clearly excluded (7, 11, 12, 15, 22, 25, 26, 28, 29, 30, 31, 32, 33, 34).

These observations may be explained by recent findings on the roles of tumoral GHRH and GHRH receptors in the growth of cancers. The fact that some cancers produce GHRH has been known for more than 2 decades (35), but recent studies also indicate that this peptide is an autocrine growth factor for many malignancies. Thus, it was shown that various cancer lines, including human endometrial, breast, ovarian, pancreatic, gastric, colorectal, lung, and bone cancers, synthesize GHRH, and their growth is stimulated by exogenous GHRH and its agonistic analogs (21, 22, 25, 27, 32, 33, 34, 36, 37). The expressions of mRNA for GHRH and GHRH peptide itself were also found in surgical specimens of human endometrial, ovarian, breast, and prostatic cancers (36, 38, 39). In three of seven endometrial cancer specimens, mRNA for GHRH was expressed at higher levels than in normal endometrium obtained from the same patients. However, this finding was not significant (40). mRNAs encoding four splice variants (SV) of GHRH receptors and specific high affinity binding sites for GHRH and its antagonistic analogs have been identified in surgical specimens of various human cancers and diverse cancer lines (25, 26, 27, 32, 33, 37, 38, 41, 42, 43, 44). The presence of GHRH receptor proteins encoded by SVs was also detected by immunohistochemistry on human endometrial cancer specimens (45). These findings suggest that the direct antiproliferative action of GHRH antagonists could be exerted through the disruption of an autocrine/paracrine stimulatory loop formed by tumoral GHRH and its tumoral receptors (22, 27, 32, 33, 37, 43, 46, 47).

Previous findings by our group suggest that GHRH might be a growth factor in endometrial cancer cells, and that its effects are exerted through SV receptors for GHRH (45). In the present study we have evaluated the effects and the mechanisms of action of a new potent antagonistic analog of GHRH, MZ-J-7-118, in the HEC-1A human endometrial cancer cell line xenografted into nude mice or cultured in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptides and reagents

GHRH antagonists MZ-J-7-118 and JV-1-42 as well as human (h) GHRH-(1–29)NH2 were synthesized in our laboratory by solid phase methods (9, 10). The chemical structure of MZ-J-7-118 is [CH3-(CH2)6-CO-Tyr1,D-Arg2,Phe(4-C1)6,Ala8,His9,Tyr(Et)10,His11,Abu15,Nle27,D-Arg28,Har29]hGHRH-(1–29)NH2, and that of JV-1-42 is [PhAc-His1,D-Arg2,Phe(4-C1)6,Arg9,Abu15,Nle27,D-Arg28,Har29]hGHRH-(1–29)NH2, where PhAc is phenylacetyl, Phe(4-C1) is 4-chlorophenylalanine, Abu is {alpha}-aminobutyric acid, Nle is norleucine, and Har is homoarginine. For daily injection, MZ-J-7-118 was dissolved in 0.1% dimethylsulfoxide in 10% aqueous propylene glycol solution (vehicle solution).

Cell lines and animals

The HEC-1A endometrial cancer cell line was established from the primary tumor of a patient with stage 1A endometrial cancer (48). It grows in athymic nude mice, forming moderately well differentiated adenocarcinomas consistent with endometrial carcinoma (grade II). HEC-1A cells are known to express the SV1 receptor for GHRH (45). This cell line was obtained from American Type Culture Collection (Manassas, VA) and was maintained in culture as previously described (45).

Five- to 6-wk-old female athymic nude mice (Ncr nu/nu) were obtained from the National Cancer Institute (Bethesda, MD). The animals were housed in sterile cages under laminar flow hoods in a temperature-controlled room with a 12-h light, 12-h dark schedule. They were fed autoclaved chow and water ad libitum.

In vivo experiments

HEC-1A human endometrial cancer cells growing exponentially were implanted into five female nude mice by sc injection of 107 cells into both flanks. Tumors resulting after 4 wk in donor animals were aseptically dissected and mechanically minced. In both experiments, 3-mm3 pieces of tumor tissue were transplanted sc into the experimental animals by a trocar needle. Tumor volume (length x width x height x 0.5236) and body weight were measured weekly. At the end of each experiment, mice were killed under anesthesia, tumors were excised and weighed, and necropsy was performed. Tumor specimens were snap-frozen and stored at –70 C. To check the toxicity, the body weights of animals were measured weekly. At the end of each experiment necropsy was performed, and organs were weighed and evaluated for macroscopic changes.

All experiments were performed in accordance with the institutional guidelines for the welfare of animals in experiments. The institutional animal care and use committee reviewed the animal care experiments and gave full approval.

In experiment 1, when HEC-1A tumors had reached a volume of approximately 38–46 mm3, mice were divided into two experimental groups of eight animals each, which received the following treatment as a daily sc injection in the dorsal region: group 1, control, vehicle solution; and group 2, MZ-J-7-118 at a dose of 10 µg/d. The experiment was terminated on d 43.

In experiment 2, after HEC-1A tumors reached a volume of 46–54 mm3, mice were divided into two groups, which received the following treatment twice daily as sc injections in the dorsal region: group 1, control, vehicle solution (12 mice); and group 2, MZ-J-7-118 at a dose of 20 µg (10 mice). The experiment was terminated on d 29.

Determinations of IGF-I, IGF-II, vascular endothelial growth factor-A (VEGF-A), and hGHRH

VEGF-A, IGF-I, and IGF-II levels were determined by RIA in extracts from tumor tissue and media from cultured cells. Tumors were homogenized in ice-cold extraction buffer (50 mM Tris-HCl, 5 mM EDTA, and 5 mM MgCl2) containing protease inhibitors. Chilled samples were stirred for 30 min, and the supernatant was obtained by centrifugation (12,000 x g, 20 min) and frozen at –70 C until use. VEGF-A, IGF-I, and IGF-II from tumor tissue and mouse IGF-I from serum were extracted using a modified acid-ethanol cryoprecipitation method (49). The method eliminates most of the binding proteins, which can interfere with the RIA. Antimouse IGF-I from PeproTech, Inc. (Rocky Hill, NJ), was used in the final dilution of 1:40,000. The standard curve was in the range of 2–2,000 fmol/tube. Total protein content was determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA).

The standard for VEGF-A was recombinant human VEGF, 38.2 kDa, consisting of 165 amino acids. Antihuman VEGF was an affinity-purified, polyclonal antibody. Both VEGF and antihuman VEGF were purchased from PeproTech, Inc. The standard was used in a range of 0.006–12.8 ng/tube. The antibody was used at a final dilution of 1:200,000. VEGF was iodinated by the lactoperoxidase method and purified by HPLC, using a reverse phase Vydac C4 column (Hesperia, CA). The assay buffer for VEGF consisted of 0.01 M sodium phosphate (pH 7.6), 0.025 M EDTA, 0.14 M NaCl, and 1% BSA. The antibody and tracer were incubated overnight at 4 C. Bound and free fractions were separated by the polyethylene glycol double-antibody method.

For measurement of hGHRH secretion by HEC-1A endometrial cancer cells in vitro, 5–6 x 106 cells were seeded in 75-cm2 flasks and allowed to attach for 24 h when McCoy’s 5A medium with 10% fetal bovine serum (FBS) medium was replaced by McCoy’s 5A medium with 5% FBS (Invitrogen Life Technologies, Inc., Carlsbad, CA). After 0, 24, and 48 h, the secretion of hGHRH into the medium and the hGHRH content of HEC-1A cells were determined by RIA as previously described (21). Inter- and intraassay variations were less that 15% and 10%, respectively.

RNA isolation and GHRH and GHRH splice variant receptor analysis

Total RNA was extracted from three control samples of HEC-1A endometrial tumor tissue, and from a sample of 34 x 106 cells of human small cell lung cancer cell line H69 (American Type Culture Collection) using the Tri-Reagent protocol (Sigma-Aldrich Corp., St. Louis, MO). Polyadenylated RNA was purified using an mRNA isolation kit (Roche, Mannheim, Germany). The yield and quality of total and mRNA were determined spectrophotometrically using 260 nm and 260/280 nm ratio, respectively. Two micrograms of RNA with a final volume of 40 µl were reverse transcribed into cDNA with the iScript cDNA synthesis kit from Bio-Rad Laboratories using an Applied Biosystems PCR system 2700 (Foster City, CA).

The expressions of GHRH and the splice variants of GHRH receptors were analyzed using the real-time PCR Bio-Rad iCycler. The gene-specific primers designed included a common area for all GHRH SV receptors (SV1, SV2, SV3, and SV4; nucleotides 48–151). For the SV receptors, the sense primer was 5'-CTACTGCCCTTAGGATGCTGG-3', the antisense primer was 5'-CCTTCCTTCTCTGGCTTCAAAG-3', and the probe was 5'-carbocyanine 5-CCCAGCTCACCACTCCTCACCCCT-black hole quencher-2–3', yielding a product of 104 bp. For the GHRH, the sense primer was 5'-ATGCAGATGCCATCTTCACCAA-3', the antisense primer was 5'-TGCTGTCTACCTGACGACCAA-3', and the probe was 5'-hexachlorofluorescein-TCTTGGTTGCTCTCTCCCTGCTGCCT-black hole quencher-1–3', amplifying a product of 150 bp. As housekeeping gene we used human ß-actin. The sense, antisense, and probe for ß-actin were 5'-CTGGAACGGTGAAGGTGACA-3', 5'-AAGGGACTTCCTGTAACAATG-3', and 5'–6-carboxyfluorescein-CAGTCGGTTGGAGCGAGCATCCCC-black hole quencher-1–3', with a product of 140 bp. All primers and probes were diluted in nuclease-free water. After taking well-factor plate readings, we evaluated the expressions of both genes. All reactions were performed using an initial denaturation step at 95 C for 3 min, followed by 45 cycles for GHRH and 40 cycles for splice variants of GHRH receptors at 95 C for 30 sec and 60 C for 1 min. As final steps we performed two cycles, one at 95 C and the other at 55 C, both for 1 min each. Bio-Rad iQ supermix was used for all PCRs, and each well contained 25 µl as the final volume, including 2 µl cDNA, 200 nM gene-specific primers, and 400 nM probes. The efficiencies of all primers (Invitrogen Life Technologies, Inc.) and probes (Integrated DNA Technologies, Coralville, IA) were tested before the experiments, and they were all efficient in the range of 95–105%.

Each sample of HEC-1A and H69 was run in triplicate for each primer. Negative samples were run for each plate consisting of no RNA into RT reaction and no cDNA into PCR. The PCR products obtained after each reaction for all genes tested were visualized in a 3% ethidium bromide-stained agarose gel.

RNA isolation from mouse liver and real-time PCR analysis

Total RNA was isolated using the Aurum Total RNA Mini Kit (Bio-Rad Laboratories). One microgram of total RNA was reverse transcribed into cDNA with 1x iScript Reaction Mix (Bio-Rad Laboratories) and 1 µl iScript reverse transcriptase (Bio-Rad Laboratories) in a total volume of 20 µl. The iCycler iQ real-time PCR detection system (Bio-Rad Laboratories) was used for sample cDNA quantification. The PCR contained 2 µl RT mixture, 300 nM gene-specific primers, and 1x iQ SYBRO Green Supermix (Bio-Rad Laboratories) in a total volume of 25 µl. The specific primer sequences for mouse IGF-I were: sense, 5'-CTGTGCCCCACTGAAGCCTA-3'; and antisense, 5'-GGACTTCTGAGTCTTGGGCATG-3'; those for ß-actin were: sense, 5'-AGATCAAGATCATTGCTCCTCCT-3'; and antisense, 5'-GGGTGTAAAACGCAGCTCAG-3'. The thermal cycling conditions comprised an initial denaturation step at 95 C for 3 min, then 40 cycles of two-step PCR, including 95 C for 15 sec and 60 C for 1 min. Data were collected during the 60 C annealing step and were further analyzed with the iCycler iQ optical system’s software. Each PCR included the five points of the calibration curve using serially diluted mouse liver cDNA. All samples were analyzed in triplicate. Negative controls included PCR amplification without cDNA and reverse transcriptase, respectively. The relative expression ratio was calculated using the mathematical model described by Pfaffl (50), with IGF-I as the target gene and ß-actin as the reference gene.

In vitro proliferation studies

Cells were seeded in 96-well microplates (Falcon, BD Biosciences, Lincoln Park, NJ) at low density corresponding to a confluence of 2–5%. After a recovery period of 24 h, the culture medium was removed and replaced with McCoy’s 5A medium with 5% FBS. The test compounds GHRH-(1–29)NH2 and MZ-J-7-118 were added at concentrations of 0.1, 1, and 10 µM. Each treatment was performed in octuplicate wells, and the experiments were repeated three times.

After 90 and 139 h, the crystal violet assay was performed to determine the OD of the wells as previously described (34). Results were calculated as %T/C, where T is the OD600nm of treated cultures, and C is the OD600nm of control cultures (medium plus vehicle).

Radioligand binding studies

A radioiodinated derivative of the GHRH antagonist JV-1-42 was prepared by the chloramine-T method as previously described (38). Preparation of the membrane fractions from HEC-1A human endometrial cancers grown in nude mice was carried out as previously reported (42). Receptor binding of GHRH was evaluated using in vitro ligand competition assays based on binding of the radiolabeled GHRH antagonist JV-1-42 to tumor membrane fractions as described in detail previously (38, 42). In brief, membrane homogenates containing 40–60 µg protein were incubated in duplicate or triplicate with 50,000–70,000 cpm [125I]JV-1-42 and increasing concentrations (10–12–10–6 mol/liter) of nonradioactive peptides as competitors in a total volume of 150 µl binding buffer. After 1 h of incubation at room temperature, the bound and the free fractions were separated by centrifugation. The supernatant was removed by aspiration, and the pellet was washed twice with ice-cold buffer, then counted in a {gamma}-counter. The Ligand-PC computerized curve-fitting software of Munson and Rodbard (51) was used to determine the type of receptor binding, dissociation constant (Kd), and the maximal binding capacity of the receptors. The receptor binding affinity of GHRH antagonist MZ-J-7-118 to GHRH receptor protein expressed on HEC-1A tumors was measured in displacement experiments based on competitive inhibition of [125I]JV-1-42 binding using various concentrations of MZ-J-7-118 (10–6–10–12 M). The IC50 value was calculated with a computerized curve-fitting program and is defined as the dose of MZ-J-7-118 that causes 50% inhibition of [125I]JV-1-42 binding.

Statistical analysis

All data are expressed as the mean ± SE The differences between mean values were evaluated by two-tailed Student’s t test. P < 0.05 was considered significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of MZ-J-7-118 on the growth of xenografts of HEC-1A human endometrial cancer

In the initial study (experiment 1), daily injection of 10 µg MZ-J-7-118 for 6 wk significantly (P < 0.05) inhibited the volume of HEC-1A tumors by 43% and tumor weight by 40% compared with controls and prolonged the tumor volume doubling time from 18.7 ± 1.4 to 25.4 ± 3.8 d (Fig. 1Go and Table 1Go). In the second experiment, MZ-J-7-118 injected twice daily at a dose of 20 µg for 4 wk significantly (P < 0.05) decreased HEC-1A tumor volume and weight by 57.9% and 50.7%, respectively (Fig. 2Go and Table 1Go). The tumor volume doubling time was significantly (P < 0.05) extended from 17.5 ± 2.8 to 36.4 ± 6.5 d in treated animals (Table 1Go). At the end of the experiment, no significant differences in body weights or in the weights or macroscopic appearance of organs were observed in treated animals compared with controls, indicating that at these doses, MZ-J-7-118 had no obvious side effects.



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  		FIG. 1. Mean tumor volumes in nude mice bearing HEC-1A human endometrial carcinoma xenografts during treatment with the GHRH antagonist MZ-J-7-118 at a daily dose of 10 µg. Vertical bars indicate the SE. *, P <0.05.

 

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  		TABLE 1. Effects of therapy with GHRH antagonist MZ-J-7-118 on the growth of HEC-1A human endometrial carcinomas xenografted into nude mice

 


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  		FIG. 2. Experiment 2: mean tumor volumes in nude mice bearing HEC-1A human endometrial carcinoma xenografts during treatment with the GHRH antagonist MZ-J-7-118 injected twice daily at a dose of 20 µg. Vertical bars indicate the SE. *, P < 0.05.

 
Effects of MZ-J-7-118 on serum levels of IGF-I and concentrations of IGF-I, IGF-II, and VEGF-A in HEC-1A endometrial tumors

Daily administration of 10 µg MZ-J-7-118 for 43 d (experiment 1) caused no significant changes in IGF-I, IGF-II, and VEGF-A levels in HEC-1 tumors compared with control animals (Table 2Go). However, serum IGF-I levels were significantly (P < 0.01) decreased by 17.7% in treated animals (Table 2Go). Twice daily injection of 20 µg MZ-J-7-118 for 29 d did not significantly alter serum IGF-I levels and tumoral IGF-II and VEGF-A levels compared with control values (Table 2Go). Surprisingly, tumoral IGF-I levels were significantly increased (P < 0.01) by 43.2% in treated mice (Table 2Go).


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  		TABLE 2. Effects of MZ-J-7-118 on serum IGF-I levels and tumoral levels of IGF-I, IGF-II, and VEGF-A as measured by RIA

 
Effects of MZ-J-7-118 on mRNA levels of IGF-I in livers of nude mice

Total RNA from livers of mice treated twice daily with 20 µg/d MZ-J-7-118 and untreated controls (experiment 2) was isolated and subjected to real-time RT-PCR analysis. PCR efficiencies were 95.6% and 101.9% for ß-actin and IGF-I, respectively. No differences in the expression level of the mRNA for IGF-I between the livers of treated and control animals could be detected, because the relative expression ratio (50) was 0.99. No significant amounts of PCR products were revealed in negative controls without cDNA or reverse transcriptase.

Presence of mRNA for GHRH and for SV of GHRH receptors in HEC-1A endometrial tumor samples

The HEC-1A tumors expressed mRNA for GHRH and for SV of GHRH receptors (Fig. 3Go). The efficiencies of GHRH SV receptors, GHRH, and ß-actin were 97.6%, 99.4%, and 95.1%, respectively. The presence of PCR products in the negative controls for all tested genes could not be detected.



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  		FIG. 3. Real-time RT-PCR analysis of GHRH splice variant receptors (A), GHRH (B), and ß-actin (C) in three samples of HEC-1A endometrial cancers (lanes 2, 3, and 4, respectively) and a sample of H69 small cell lung cancer cell line as a positive control (lane 6). The DNA molecular weight marker is shown in lane 1. All PCRs yielded products of the expected sizes of 104 bp for GHRH SV receptors, 150 bp for GHRH, and 140 bp for ß-actin. The negative controls with no RNA in the RT reaction and no cDNA in the PCR are shown in lanes 5 and 7, respectively.

 
Receptor binding studies

In membranes of untreated HEC-1A endometrial tumors, radiolabeled JV-1-42 was bound to a single class of high affinity binding sites with a mean Kd of 2.28 ± 0.37 nM and a mean maximal binding capacity of 206.6 ± 12.7 fmol/mg protein (Fig. 4Go). The concentration of the GHRH antagonist MZ-J-7-118 required to inhibit the binding of [125I]JV-1-42 by 50% (IC50) was 0.13 ± 0.04 nM, indicating a high affinity of MZ-J-7-118 to GHRH binding sites expressed on HEC-1A tumors.



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  		FIG. 4. Representative example of Scatchard plots of [125]JV-1-42 binding to a membrane fraction isolated from HEC-1A human endometrial cancer. Specific binding was determined as previously described (38 42 ). Each point represents the mean of triplicate determinations.

 
Production and secretion of hGHRH by HEC-1A endometrial cells cultured in vitro

The hGHRH content of HEC-1A cells was measured by RIA and found to be in the range of 1805–5180 pg/mg protein in cell homogenates from different batches of cell cultures. The concentration of hGHRH in culture medium was also determined by RIA at different time intervals. Significant amounts of secreted hGHRH were found in medium from cells after 24 and 48 h of culture, whereas medium without cells did not contain detectable levels of hGHRH (Table 3Go).


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  		TABLE 3. Production of GHRH in culture medium from HEC-1A human endometrial cancer cells

 
Effect of GHRH-(1–29)NH2 and the GHRH antagonist MZ-J-7-118 on the proliferation of HEC-A endometrial cells in vitro

HEC-1A cells cultured in vitro were exposed to various concentrations of GHRH-(1–29)NH2 and MZ-J-7-118, and the effect on the proliferation was determined by crystal violet assay. GHRH-(1–29)NH2 at 0.1–10 µM did not affect the growth of HEC-1A cells (data not shown). The GHRH antagonist MZ-J-7-118 did not significantly inhibit the proliferation of HEC-1A cells at the concentration of 0.1 µM, but a significant inhibition of 15% (P < 0.05) was observed using 1 µM. MZ-J-7-118 at a concentration of 10 µM significantly inhibited (P < 0.01) the growth of HEC-1A cells by 42.8%. The results of a representative experiment are shown in Fig. 5Go.



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  		FIG. 5. Effect of the GHRH antagonist MZ-J-7-118 on the proliferation of the HEC-1A human endometrial cancer cell line. This experiment is representative of three independent experiments, each performed in octuplicate wells. The relative cell numbers in treated and control wells were determined by crystal violet staining and expressed as percent T/C values, where T is the absorbance of treated cultures, and C is the absorbance of control cultures. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Novel approaches to therapies for various cancers could be based on the use of peptide analogs that block the endocrine and autocrine/paracrine stimulatory pathways promoting tumor growth (7, 8). GHRH antagonists belong to this class of peptide antitumor agents and were initially developed for treatment of cancers dependent on hepatic production of IGF-I (19). GHRH antagonists can suppress the secretion of GH from the pituitary by inhibiting (blocking) the binding of hypothalamic GHRH to pituitary GHRH receptors (7, 8). This results in a decrease in production of hepatic IGF-I, leading to an inhibition of the growth of IGF-I dependent tumors (7, 8). Nevertheless, subsequent evidence pointed toward a major role of local GHRH and tumoral GHRH receptors in the pathogenesis of various cancers. Thus, more recent findings indicate that most of the antitumor effects of the GHRH antagonists could be mediated through a direct action on the cancer cells (7, 22, 27, 32, 33, 37, 38, 41, 43, 46, 47). It has been shown that GHRH antagonists increase the apoptosis and decrease the proliferation rate of MXT mouse mammary cancers, HT-29 human colon cancers, and SW-1990 human pancreatic cancers in vivo (11, 12, 29). Although attempts to detect the presence of pituitary-type GHRH receptors on various cancers were unsuccessful (15, 26, 31, 41, 52), we were able to demonstrate by the isolation and sequencing of corresponding cDNAs that these tumors contained SVs of GHRH receptors. These SVs of GHRH receptors on tumors might mediate the direct effects of GHRH and its antagonists (41, 42) and are distinct from the pituitary-type GHRH receptors or receptors for other peptides of the secretin/glucagon family (41). All of the SVs of the GHRH receptor have a retained intronic sequence at the 5' end, but lack the first three exons (41, 42). The lack of the first three exons in SV1 might result in a tumoral receptor protein, in which most of the large NH2-terminal extracellular domain, characteristic of the pituitary receptor, is truncated (41, 42). Thus, receptor SV1 appears to encode a functional receptor protein with binding affinity for GHRH and its analogs different from that of the pituitary GHRH receptor (42). The participation of SV1 in cell proliferation signaling has been shown by transfection in NIH-3T3 mouse fibroblast cells (47). In this study, transfection of 3T3 cells with SV1 produced specific high affinity binding sites for radiolabeled GHRH antagonist JV-1-42 and conferred a greatly augmented sensitivity to GHRH analogs. Thus, the expression of SV1 increased the mitogenic responses to GHRH or the GHRH agonist JI-38 and the antimitogenic response to the GHRH antagonist JV-1-38 compared with control cells that did not express SV1 (47).

It has been reported that locally produced GHRH is present in appreciable quantities in specimens of endometrial cancer as well as in HEC-1A human endometrial cancer xenografts (36). The expression of GHRH was found in 16 of 22 surgical specimens (77%) of endometrial cancers (36). It was also demonstrated that 43% of human endometrial cancers and HEC-1A human endometrial cancer cells expressed the SV1 receptor. The proliferation of HEC-1A cells was stimulated by GHRH peptide (45). Ablation of the SV1 receptor in this cell line by an antisense RNA-based approach reduced the rate of cell proliferation in the absence of exogenous GHRH and decreased the sensitivity of cells to exogenous GHRH (45).

The current study reports the presence of specific, high affinity receptors for GHRH antagonists on HEC-1A tumor membranes and the expression of mRNA for GHRH and for SV receptors by the tumors. We also found that cultured HEC-1A cells produce and secrete GHRH into the culture medium and that their proliferation is significantly inhibited by the GHRH antagonist MZ-J-7-118. However, GHRH failed to stimulate the growth of HEC-1A cells in our experiments. This finding could be explained by a high endogenous secretion of GHRH by HEC-1A cells, which induces maximal stimulation of cell growth, so that no additional growth-promoting effect could be achieved by exogenous administration of GHRH.

High concentrations of the GHRH antagonist MZ-J-7-118, in the range of 1–10 µM, were necessary to inhibit cell proliferation in vitro, as in our previous results with other cancer lines and transfected cells expressing the tumoral SV receptors for GHRH (11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23, 25, 26, 28, 29, 30, 31, 32, 33, 34, 37, 47). However, GHRH antagonists, including MZ-J-7-118, displayed nanomolar or subnanomolar binding affinities to the tumoral GHRH receptors (21, 26, 27, 28, 38, 41, 42, 45, 47) and effectively inhibited the growth of various tumors in vivo, even though their concentrations in the sera of nude mice reached peak levels of only about 40 nM after sc injection of a dose of 40 µg (19). Similar findings were observed with other peptide hormone analogs, such as agonists or antagonists of LHRH, antagonists of bombesin/gastrin-releasing peptide or vasoactive intestinal polypeptide, or somatostatin analogs, which were also much less effective in vitro than in vivo (7, 25, 26, 31, 34). Large differences between the concentrations of peptide hormones that affect cancer cell proliferation in vitro and in vivo could be related to the different growth characteristics of the cells under these conditions. Thus, the doubling time of HEC-1A tumors in vivo is about 18 d, as opposed to only 24–48 h for the cells cultured in vitro. The increased availability of nutrients provided by the serum used in the cell culture medium and the lack of contact inhibition during the proliferation experiment in vitro could account for the much more aggressive growth characteristics of the cells in vitro and their reduced sensitivity to the peptide hormonal agents that affect cell proliferation. The absence of stimulatory effects of GHRH in our cell proliferation studies could also be due to the experimental conditions, including the presence of 5% FBS in the culture medium.

Our in vitro findings are supported by the results in vivo. The antagonist MZ-J-7-118 at a dose of 10 µg/d significantly decreased tumor growth by about 40%. Administration of 40 µg/d MZ-J-7-118 significantly decreased tumor volume and weight by more than 50% and significantly prolonged tumor doubling time. No obvious toxic side effects of MZ-J-7-118 were observed. To assess whether the endocrine actions of MZ-J-7-118 contributed to the antitumor effect in vivo, we measured serum levels of IGF-I and determined the IGF-I content in livers of treated animals. In mice that received 10 µg/d MZ-J-7-118, a moderate decrease in the serum IGF-I level of 17% was observed. Although the effect was significant, this result should be interpreted with caution, because it is based on a relatively small sample size of eight animals per group. In the second experiment, based on 10–12 animals/group, IGF-I levels in serum or liver were not decreased after treatment with a larger dose of 40 µg/d MZ-J-7-118. Several recent studies with other tumors xenografted into nude mice also showed that the antagonist MZ-J-7-118 given at doses of 20–40 µg/d did not inhibit serum IGF-I (Engel, J. B., G. Keller, A. V. Schally, G. Halmos, M. Zarandi, and J. L. Varga, unpublished observations). The proliferation of HEC-1A cells is stimulated by IGF-I (24); therefore, a reduction in the levels of serum IGF-I could participate in the antitumor mechanism of the GHRH antagonists. MZ-J-7-118 at the doses used in our study apparently had no clear endocrine inhibitory effect on the hypothalamic GHRH-pituitary GH-hepatic IGF-I axis, although the growth of endometrial tumors was dose-dependently inhibited. Consequently, it appears that the suppression of serum IGF-I did not play a major role in the antiproliferative effect of this antagonist on HEC-1A tumors in vivo.

Because tumoral IGF-I, IGF-II, and VEGF-A levels were decreased by GHRH antagonists in some tumor models (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 27, 29, 30, 34, 46, 52, 53, 54), we compared these growth factors in treated and control tumor samples to elucidate the mechanism of action of MZ-J-7-118 in HEC-1A tumors. At a dose of 10 µg/d, MZ-J-7-118 did not significantly influence tumoral levels of IGF-I, IGF-II, and VEGF-A. When administered at 40 µg/d, MZ-J-7-118 did not significantly alter tumoral IGF-II and VEGF-A, but paradoxically, IGF-I levels in tumors were elevated in treated animals. Thus, in this model, the antiproliferative effect of MZ-J-7-118 is not linked to inhibition of the tumoral concentrations of IGF-I, IGF-II, and VEGF-A. However, it should be mentioned that various components of the tumoral IGF system not investigated in this study, such as the expression of tumoral IGF-I gene, IGF receptors I and II (IGFR-I and -II), and IGF-binding proteins (IGFBPs), could also be affected by the GHRH antagonist. The observed increase in tumoral IGF-I concentrations after treatment with 40 µg/d MZ-J-7-118 in the absence of a significant effect on serum IGF-I might indicate a regulatory action on the tumoral IGF system. An increase in the expression of the tumoral IGF-I gene or increases in the expression levels and tumoral concentrations of IGFR and various types of IGFBPs could occur by negative feedback mechanisms and contribute to the elevation of tumoral IGF-I levels. It has been shown previously that the GHRH antagonists MZ-4-71 and MZ-5-156 decrease mRNA expression for IGF-II in prostatic, mammary, ovarian, colorectal, and pancreatic cancers; glioblastomas; and osteosarcomas, but increase the mRNA expression of IGFR-II in human experimental glioblastomas and ovarian carcinomas (18, 26). IGFR-II and IGFBPs, especially IGFBP-3, can act as tumor suppressors by reducing the concentrations of free IGFs or by IGF-independent mechanisms (18, 26, 55)

GHRH appears to fulfill some characteristics of an autocrine growth factor in the HEC-1A endometrial cancer cell line, because it is expressed and secreted by the tumor, and an isoform of the GHRH receptor is also present on tumors. MZ-J-7-118 shows high binding affinity, in the subnanomolar range, to HEC-1A tumor membranes, and it can counteract the effect of locally produced GHRH and inhibit the growth of HEC-1A cells in vitro and in vivo.

In conclusion, this work extends previous in vitro findings and corroborates an involvement of local GHRH and its tumoral receptors in the pathophysiological regulation of endometrial cancer (36, 45). Our studies demonstrate that GHRH antagonists can slow the growth of HEC-1A human endometrial cancer xenografted into nude mice. Additional studies in a number of endometrial cancer cell lines or primary tumors derived from patients with advanced disease should be performed to support these findings. In view of the possible function of GHRH as an autocrine growth factor in endometrial cancer and the significantly slower growth of HEC-1A tumors after therapy with MZ-J-7-118, GHRH antagonists could be considered for the development of a new adjuvant therapy for endometrial cancer.


   Acknowledgments
 
The work is dedicated to the late Ana Maria Comaru-Schally, M.D., F.A.C.P., who died recently from thyroid cancer, for her intellectual, spiritual, and personal contribution to this work and for the inspiration she provided for this project.


   Footnotes
 
This work was supported by the medical research service of the Department of Veterans Affairs and a grant from ZENTARIS Gmbh (Frankfurt am Main, Germany) to Tulane University (all to A.V.S.). G.K. is supported by a fellowship within the postdoctorate program of the German Academic Exchange Service.

First Published Online March 22, 2005

Abbreviations: FBS, Fetal bovine serum; h, human; IGFBP, IGF-binding protein; IGFR, IGF receptor; SV, splice variant; VEGF, vascular endothelial growth factor.

Received November 4, 2004.

Accepted March 15, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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