Cancer Research Landon Prizes for Basic and Translational Cancer Research Tumor Immunology: New Perspectives
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Szereday, Z.
Right arrow Articles by Busto, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Szereday, Z.
Right arrow Articles by Busto, R.
[Cancer Research 63, 7913-7919, November 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Antagonists of Growth Hormone-Releasing Hormone Inhibit the Proliferation of Experimental Non-Small Cell Lung Carcinoma

Zoltan Szereday1,2, Andrew V. Schally1,2, Jozsef L. Varga1,2, Celia A. Kanashiro1,2, Francine Hebert1, Patricia Armatis1, Kate Groot1, Karoly Szepeshazi1,2, Gabor Halmos1,2 and Rebeca Busto1,2

1 Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center,
2 Section of Experimental Medicine, Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies show that antagonists of growth hormone-releasing hormone (GH-RH) inhibit proliferation of various cancers indirectly through blockage of the endocrine GH-insulin-like growth factor (IGF) I axis and directly by an action on tumor cells involving the suppression of autocrine/paracrine IGF-I, IGF-II, or GH-RH. The effectiveness of therapy with GH-RH antagonist JV-1–38 and its mechanisms of action were investigated in NCI-H838 non-small cell lung carcinoma (NSCLC) xenografted s.c. into nude mice and in vitro. Treatment with GH-RH antagonist JV-1–38 significantly (P < 0.05–0.001) inhibited tumor growth as demonstrated by a 58% decrease in final tumor volume, 54% reduction in tumor weight, and the extension of tumor-doubling time from 8.5 ± 1.38 to 12 ± 1.07 days as compared with controls. Using ligand competition assays with 125I-labeled GH-RH antagonist JV-1–42, specific high-affinity binding sites for GH-RH were found on tumor membranes. Reverse transcription-PCR revealed the expression of mRNA for GH-RH and splice variant 1 (SV1) of GH-RH receptor in H838 tumors. Reverse transcription-PCR analysis also demonstrated that H838 tumors express IGF-I and IGF-I receptors. Tumoral concentration of IGF-I and its mRNA expression were significantly decreased by 25% (P = 0.05) and 65% (P < 0.001), respectively, in animals receiving JV-1–38, whereas serum IGF-I levels remained unchanged. In vitro studies showed that H838 cells secreted GH-RH and IGF-I into the medium. The growth of tumor cells in vitro was stimulated by IGF-I and inhibited by GH-RH antagonist JV-1–38 and a GH-RH antiserum. Our results extend the findings on the involvement of IGF-I in NSCLC and suggest that GH-RH may be an autocrine growth factor for H838 NSCLC. The antitumorigenic action of GH-RH antagonists could be partly direct and mediated by SV1 of tumoral GH-RH receptors. The finding of GH-RH and SV1 of GH-RH receptors in NSCLC provides a new approach to the treatment of this malignancy based on the use of antagonistic analogues of GH-RH.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lung cancer is the leading cause of cancer-related deaths in the Western world (1) . About 155,000 Americans die of lung tumors each year. This represents 25–30% of all cancer deaths in the United States (2) . The estimated annual number of lung cancer deaths worldwide is nearly 1 million (3) . Consequently, it is necessary to explore novel therapeutic approaches to lung carcinomas.

NSCLC1 account for ~80% of all lung cancer cases (4) . NSCLC is considered an aggressive tumor type with poor prognosis that typically develops resistance to the various treatment modalities (5 , 6) .

Several series of antagonistic analogues of GH-RH were synthesized in our laboratory in the past 10 years in an endeavor to develop a new class of antitumor agents (7, 8, 9) . We have shown that GH-RH antagonists inhibit the growth of diverse cancers such as pancreatic (10) , colorectal (11) , prostatic (12 , 13) , breast (14) , and renal (15) cancers; glioblastomas (16) ; osteosarcomas (17 , 18) ; SCLC; and NSCLC (19 , 20) . GH-RH antagonists block the secretion of GH from the pituitary by inhibiting the binding of hypothalamic GH-RH to pituitary GH-RH receptors. Thus, the antineoplastic effect of GH-RH antagonists is caused part by the suppression of the GH release from the pituitary, which in turn results in the inhibition of the hepatic production of IGF-I (8 , 9) . An inhibition of experimental SCLC and NSCLC cancers, achieved with relatively high doses of early GH-RH antagonists, was associated with reduced serum IGF-I levels (20) . IGF-I is a well-known mitogen for various cancers including NSCLC (21 , 22) . In addition, it was observed that GH-RH antagonists can inhibit the proliferation of various cancer lines including H-69 human SCLC by a direct action in vitro, under conditions in which the contribution of the hypothalamic GH-RH/pituitary growth hormone/hepatic IGF-I axis is clearly excluded (9 , 14 , 19 , 23, 24, 25, 26, 27) . This finding may be accounted for by the presence of GH-RH and GH-RH receptors on tumors. Thus, the endogenous production of GH-RH was detected in human SCLC (19) , prostate cancer (28) , N-methyl-N'-nitro-N-nitrosoguanidine/human osteosarcoma, and SK-ES-1 Ewing sarcoma (27) cell lines. The presence of mRNA for GH-RH was also demonstrated in cell lines of prostatic, breast, ovarian, and endometrial cancers; SCLC; osteosarcomas; Ewing sarcomas (19 , 27 , 29 , 30) ; and specimens of human prostatic, breast, ovarian, and endometrial cancers (30, 31, 32) . The proliferation of some cancer lines such as H-69 SCLC, N-methyl-N'-nitro-N-nitrosoguanidine/human osteosarcomas, SK-ES-1 Ewing sarcomas could be stimulated by administration of GH-RH and its agonistic analogues or, conversely, inhibited by GH-RH antagonists (19 , 27) . SVs of GH-RH receptors, which could mediate the direct effects of tumoral GH-RH and its synthetic antagonists, have been recently identified on various human cancers (33 , 34) . These observations suggest that GH-RH may be an autocrine growth factor for some cancers, and the direct antiproliferative action of GH-RH antagonists could be exerted through the disruption of an autocrine/paracrine-stimulatory loop (19 , 27, 28, 29 , 35) .

In contrast to the extensive findings available on other cancers, no studies have been reported thus far regarding the possible involvement of GH-RH and its tumoral receptors in the pathogenesis of NSCLC. Such investigations could open new venues toward the management of this malignancy, which represents a major national and international health problem. In this study, we have evaluated the effects and the mechanism of action of a new potent antagonistic analogue of GH-RH, JV-1–38, in NCI-H838 human NSCLC cell line. This antagonist, JV-1–38, was selected for the present study because in previous oncological investigations in prostate and breast cancers, it appeared to be the most potent among the analogues tested (8 , 9) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Peptides.
hGH-RH1–29NH2 and GH-RH antagonist JV-1–38 [PhAc-Tyr1, D-Arg2, Phe(4-Cl)6, Har9, Tyr(Me)10, Abu15, Nle27, Har29] human GH-RH1–29NH2 were synthesized in our laboratory by solid phase methods as described previously (7) . For daily injections, the peptide was dissolved in 0.1% DMSO in sterile aqueous 10% propylene glycol (vehicle solution).

Cell Line, Animals, and Tumors.
Human NSCLC cell line NCI-H838, obtained from American Type Culture Collection (Manassas, VA), was derived from a primary adenocarcinoma of the lungs of a 59-year-old Caucasian man who was a smoker. The tumor was metastatic to the lymph nodes. This cell line was maintained in culture using DMEM with 100 units/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, 2 mM-glutamine (all from Invitrogen, Carlsbad, CA), and 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA).

Male athymic (Ncr-nu/nu) nude mice, ~6 weeks old on arrival, were obtained from the National Cancer Institute (Frederick Cancer Research and Development Center, Frederick, MD), housed in laminar air-flow cabinets under pathogen-free conditions with a 12-h light/12-h dark schedule, and fed autoclaved standard chow and water ad libitum. Xenografts were initiated by s.c. injection of 15 x 106 H838 cells into the right flanks of five male nude mice. Tumors resulting after 5 weeks were aseptically dissected and mechanically minced, and 3-mm3 pieces of tumor tissue were transplanted s.c. with a trocar needle. All experiments were conducted in accordance with institutional guidelines for animal care.

In Vivo Studies.
The experiment was started when tumors had grown to ~35 mm3. Animals were randomly divided into two treatment groups: group 1 (seven mice), control, vehicle solution; and group 2 (eight mice), GH-RH antagonist JV-1–38 injected s.c. twice a day at a dose of 10 µg/animal. Tumor volume (length x width x height x 0.5236) and body weights were measured twice a week. The experiment was terminated on day 30. Mice were sacrificed, and tumors were dissected, cleaned, and weighed. Tumor specimens were snap-frozen and stored at -70°C for various analyses. Tumor volume-doubling time was calculated between day 1 and day 30 using the formula

as described by Smolev et al. (36) .

Histological Methods.
The methods used in the histological investigations were described (10) , including the detection of the NOR in tumor cell nuclei. The argyrophilic NOR count is a good indicator of cell proliferation rate.

In Vitro Studies.
The effect of various agents on the rate of cell proliferation in SFM was evaluated by the crystal violet assay described previously (37) . Briefly, cells were seeded into 96-well microplates and cultured for 18 h; then the medium was removed and replaced by N2E medium containing the test compounds dissolved in 0.1% DMSO (all medium components were from Life Technologies, Inc., Gaithersburg, MD, or Sigma-Aldrich, St. Louis, MO). Test compounds included IGF-I (from Life Technologies, Inc.), JV-1–38, GH-RH, and a rabbit polyclonal antiserum raised against human GH-RH1–40 (SV-95; see "RIA for GH-RH, IGF-I, and IGF-II"), in octuplicate wells each. Controls received only DMSO in N2E medium. After 3–5 days of culture, when the cells approached the end of the exponential phase of growth, growth was stopped using 1% glutaraldehyde. The fixed plates were stored in PBS until staining with 0.02% crystal violet. Results were calculated as %T/C, where T = absorbance of treated cultures and C = absorbance of control cultures x 100.

The effects of JV-1–38 (10 µM) on the expression of mRNA for IGF-I and IGF-II were also measured. The culture medium from H838 cells grown in culture flasks (quadruplicate flasks, 10 million cells in each) was replaced by N2E medium containing 10 µM JV-1–38 or by N2E medium alone in the case of controls. After 48 h, the cells were harvested, total RNA was extracted from control and treated cells, and the expression of mRNA for IGF-I and IGF-II was measured as described below.

The measurement of incorporation of [methyl-3H]thymidine into H838 NSCLC cells was also used as a quantitative approach to determine the effects of GH-RH, GH-RH antagonist JV-1–38, IGF-I, and the GH-RH antiserum SV-95. Cells were seeded into 96-well Falcon microplates at 5 x 103 cells/well and cultured in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin G (sodium salt; 100 units/ml), and streptomycin sulfate (100 µg/ml) in a humidified atmosphere containing 5% CO2 at 37°C. After 2 days, the confluent cultures were washed with N2E medium containing 0.3% BSA and maintained in this medium for 2 additional days. The compounds were then added to the cultured cells, incubated for a period of 20 h, and pulsed with 0.25 µCi/well [methyl-3H]thymidine (specific activity, 25 Ci/mmol; Amersham Corp.) in a total volume of 175 µl/well for 3–4 h. The cells were fixed with ice-cold 10% trichloroacetic acid, washed twice with trichloroacetic acid, and solubilized in 0.2 N NaOH. The lysate was mixed with scintillation mixture (CytoScint; ICN Biomedicals), and the radioactivity was determined by liquid scintillation counting (LS 380 I; Beckman Instruments, Irvine, CA).

RIAs for GH-RH, IGF-I, and IGF-II.
The methods used for determination of IGF-I levels in serum as well as IGF-I and IGF-II concentrations in the cytosol fraction of tumors, after acid-ethanol cryoprecipitation have been described in detail (13 , 17 , 18 , 20) .

For the measurement of GH-RH, IGF-I, and IGF-II secretion by H838 NSCLC cell line in vitro, 5–6 x 106 cells were seeded in 75-cm2 flasks and allowed to attach for 24 h when the medium was replaced by serum-free N2E medium. After 0, 24, or 48 h in culture, 10 ml of medium from H838 NSCLC cells were lyophilized and reconstituted in 1 ml of PBS-EDTA buffer containing 1% BSA, 1 mM DTT, and 0.001% pepstatin A. IGF-I and IGF-II in these samples were assayed directly without an acid-ethanol cryoprecipitation step. GH-RH was measured by using 125I-GH-RH1–40 (Bachem) as labeled hormone and anti-GH-RH1–40 (SV-95) antiserum generated in our laboratory at the final dilution of 1:60,000, which cross-reacts 100% with GH-RH1–29NH2, as described earlier (38) . GH-RH1–29NH2 was used as a standard, in the range of 0.0125–25 ng/tube. Intraassay and interassay coefficients of variation were <10% and 15%, respectively.

RNA Extraction.
Total RNA was extracted from frozen tissue samples using the Micro RNA Isolation Kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. The quality and quantity of RNAs were assessed by spectrophotometry at 260 and 280 nm (A260/A280 >1.8) before reverse transcription. RNA to be analyzed for GH-RH was subjected to further isolation of poly(A)RNA using the MicroPoly(A) Pure Small Scale mRNA Purification Kit (Ambion, Austin, TX) according to the manufacturer’s instructions.

Reverse Transcription.
Reverse transcription was performed using the GeneAmp RNA Core Kit (Perkin-Elmer, Foster City, CA). Two µg of total RNA (for IGF-I, IGF-II, IGF-IR, and ß-actin) or 4 µg of total RNA (for GH-RH receptors) or 2 µg of poly(A+)RNA (for GH-RH) were reverse transcribed into cDNA with Moloney murine leukemia virus reverse transcriptase according to the manufacturer’s instructions.

PCR Amplification.
The PCR amplifications of the cDNAs for human ß-actin, IGF-I, IGF-IR, and GH-RH receptors were performed using the GeneAmp RNA Core Kit (PerkinElmer), and those for IGF-II and GH-RH were performed using the GeneAmp Gold RNA PCR Reagent Kit (PerkinElmer) with 3 µl of cDNA (for IGF-I and ß-actin), 14 µl of cDNA (for GH-RH), 2.5 µl of cDNA (for IGF-II and IGF-IR), or 5 µl of cDNA (for GH-RH receptors) was amplified in a 25-µl solution containing 2 mM MgCl2 (for IGF-I, GH-RH receptors, IGF-IR, and ß-actin) or 2.5 mM MgCl2 (IGF-II and GH-RH), 1x PCR buffer, 200 µM concentrations of each deoxynucleotide triphosphate, 2.5 units of AmpliTaq DNA polymerase (for IGF-I, IGF-IR, GH-RH receptors, and ß-actin), 1 unit of AmpliTaq Gold DNA Polymerase and 0.2 µM concentrations of each primer (for IGF-I and ß-actin), 0.15 µM concentrations of each primer (for IGF-II and IGF-IR), 0.75 µM concentrations of each primer (GH-RH), or 0.5 µM concentrations of each primer (GH-RH receptors). The primers used were 5'-ATC TGG CAC CAC ACC TTC TAC AA TGA GCT GCG-3' and 5'-GCA GCG GAA CCG CTC ACC GCC AAT GGT GAT-3' for human ß-actin; 5'-ACA TCT CCC ATC TCT CTG GAT TTC CTT TTG C-3' and 5'-CCC TCT ACT TGC GTT CTT CAA ATG TAC TTC C-3' for IGF-I; 5'-AGT CGA TGC TGG TGC TTC TCA CCT TCTT GGC-3' and 5'-TGC GGC AGT TTT GCT CAC TTC CGA TTG CTG G-3' for IGF-II; 5'-AAC CAC GAG GCT GAG AAG CT-3' and 5'-CAG CAT AAT CAC CAA CCC TC-3' for IGF-IR; 5'-ATT TGA GCA GTG CCT CGG AG-3' and 5'-TTT GTT CTG CCC ACA TGC TG-3' for GH-RH; 5'-CCT ACT GCC CTT AGG ATG CTG G-3' and 5'-ATC TCA CGG AAG TGG CAT GGC C-3' for the first PCR; and 5'-GCA CCT TTG AAG CCA GAG AAG G-3' and 5'-CAC GTG CCA GTG AAG AGC ACG G-3' for the second PCR for GH-RH receptors. For ß-actin, samples were denatured for 1 min at 94°C and then subjected to 28 cycles of 94°C for 45 s, 60°C for 45 s, and then 72°C for 45 s with a final extension of 10 min at 72°C. For IGF-I, samples were denatured at 97°C for 100 s, then subjected to 5 cycles of 97°C for 20 s, 60°C for 30 s, and 72°C for 30 s, and finally 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s with a final extension of 7 min at 72°C. For IGF-II, samples were denatured at 95°C for 10 min and the subjected to 35 cycles of 94°C for 40 s and 60°C for 30 s with a final extension of 72°C for 7 min. For IGF-IR, samples were subjected to one cycle of 95°C for 3 min, 56°C for 1 min, and 72°C for 1 min and then 30 cycle of 95°C for 35 s, 56°C for 35 s, and 72°C for 40 s, with a final extension of 7 min at 72°C. For GH-RH, samples were denatured at 95°C for 10 min and subjected to 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s with a final extension of 7 min at 72°C. For GH-RH receptors, samples were denatured at 95°C for 3 min followed by 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 2 min with a final extension of 7 min at 72°C; a secondary PCR of 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min with a final extension of 7 min at 72°C was subsequently carried out on 1 µl of the primary PCR product. The number of cycles was determined in preliminary experiments to be within the exponential range of PCR amplification. Negative controls were run in parallel to exclude genomic DNA contamination. PCR products were electrophoresed on a 1.8% agarose gel, stained with ethidium bromide and visualized under UV light. PCR products were quantitated using the Kodak EDAS 290 imaging system with Kodak 1D Image Analysis Software. Experiments were performed in duplicate and relative mRNA levels of each gene were normalized versus the corresponding levels of human ß-actin.

Radioligand Binding Studies.
Radioiodinated derivative of GH-RH antagonist JV-1–42 was prepared by the chloramine-T method as described (31) . Preparation of the membrane fractions from H-838 human non-SCLCs grown in nude mice was conducted as reported (34) . Receptor binding of GH-RH was performed using in vitro ligand competition assays based on the binding of radiolabeled JV-1–42 to tumor membrane fractions described in detail previously (34) . The characteristics of the specific ligand binding were determined with the Ligand-PC curve-fitting software and by Scatchard analysis.

Statistical Analysis.
The SigmaStat Software was used for the statistical analysis of data. Data are presented as mean ± SE. Differences between the groups of the in vivo study were evaluated by one way ANOVA and the Fisher or Mann-Whitney methods. The results of in vitro experiments were compared by one way ANOVA and the Student-Newman-Keuls method.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of JV-1–38 on Growth of Xenografts of H838 NSCLC.
Four weeks after the initiation of the treatment, the tumor volume in the animals given JV-1–38 was significantly (P < 0.001) decreased to 214.65 ± 42.86 mm3, as compared with controls, which measured 509.04 ± 124.48 mm3 (Fig. 1Citation ; Table 1Citation ) and which corresponded to 58% growth inhibition (Table 1)Citation . The weight of treated tumors (219 ± 39 mg) was also 54% lower compared with that of the controls (479 ± 146 mg; P < 0.05). Treatment with JV-1–38 significantly prolonged the tumor-doubling time from 8.5 ± 1.38 to 12 ± 1.07 days (P < 0.05; Table 1Citation ). At the end of the experiment, no significant differences in body weights and the weights of various organs were observed between the groups, indicating that treatment with JV-1–38 was not toxic to the tumor-bearing animals (data not shown).



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 1. Changes in tumor volume in athymic nude mice bearing s.c. transplanted H838 NSCLC during treatment with GH-RH antagonist JV-1–38 administered by s.c. injections at a dose of 10 µg/animal twice a day (controls, n = 7; treated, n = 8). Bars, SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 

View this table:
[in this window]
[in a new window]
  		Table 1 Effect of treatment with GH-RH antagonist JV-1–38 on tumor volume, tumor weight, and tumor-doubling time of H838 human NSCLC grown in nude mice

 
Histology.
Histologically, no differences in proliferation characteristics were detected between treated and control tumors. Although the apoptotic index was ~47% higher and the argyrophilic NOR counts lower by 10% in the treated group, the differences were not significant statistically, because only a few tumor samples were analyzed histologically.

Effects of JV-1–38 on the Serum Levels of IGF-I, the Concentrations of IGF-I and IGF-II in H838 Tumors, and the Expression of mRNA for IGF-I and IGF-II.
No significant differences were found in the serum levels of IGF-I in nude mice bearing H838 NSCLC and treated with GH-RH antagonist JV-1–38 (298.5 ± 25.7 ng/ml), as compared with the controls (261.9 ± 24.0 ng/ml; Table 2Citation ). The concentration of IGF-I in control tumor samples was 170.3 ± 14.1 pg/100 µg of protein, and IGF-II was not detectable. Therapy with JV-1–38 significantly decreased the content of IGF-I in tumors to 127.5 ± 6.5 pg/100 µg of protein (25% reduction, P = 0.05; Table 2Citation ). Densitometric analysis of the PCR products corresponding to mRNA for IGF-I (Fig. 2A)Citation showed that the expression of IGF-I gene in H838 tumors of animals treated with the GH-RH antagonist was reduced by 65% (P < 0.001) as compared with the controls, which were accepted 100% (Table 2)Citation . The expression of IGF-II gene was found in tumor samples, but the level of mRNA for IGF-II did not change after the therapy with JV-1–38 (data not shown). Tumor samples also expressed mRNA for IGF-I receptor (Fig. 2B)Citation .


View this table:
[in this window]
[in a new window]
  		Table 2 Effect of treatment with GH-RH antagonist JV-1–38 on the serum levels of IGF-I in nude mice and the concentrations of IGF-I protein and mRNA in xenografts of H838 human NSCLC

 


View larger version (44K):
[in this window]
[in a new window]
  		Fig. 2. A, analysis of the expression of mRNA for IGF-I and human ß-actin in control (Lanes 1–7) and JV-1–38-treated (Lanes 8–15) H838 human NSCLC tumors grown in nude mice. Products of the expected sizes of 514 bp (IGF-I) and 459 bp (human ß-actin) were found in H838 tumors. Lane M, 100-bp DNA molecular weight marker. B, expression of mRNA for IGF-IR in H838 human NSCLC tumors grown in nude mice. Products of the expected size of 447 bp were found in control tumors (Lanes 1–7). Lane M, 100-bp DNA molecular weight marker.

 
In cultures, 48-h exposure of cell line to 10 µM JV-1–38 did not significantly affect the levels of mRNA for IGF-I and IGF-II, as compared with controls (data not shown).

Secretion of GH-RH, IGF-I, and IGF-II by H838 Cells Cultured in Vitro.
The concentrations of GH-RH, IGF-I, and IGF-II in the SFM were measured by RIA. Significant amounts of secreted GH-RH and IGF-I were found in medium from cells after 24 and 48 h of culture as shown in Table 3Citation , whereas the SFM without cells did not contain detectable levels of GH-RH and IGF-I. IGF-II was not detectable.


View this table:
[in this window]
[in a new window]
  		Table 3 Production of GH-RH and IGF-I in culture medium from H838 human NSCLC cells

Aliquots of medium at the indicated periods of time were subjected to RIA for the detection of GH-RH and IGF-I. The results are representative of three independent experiments.

 
Expression of mRNA for GH-RH in H838 NSCLC Tumors.
Polyadenylated RNAs isolated from tumor samples were subjected to RT-PCR analysis for the expression of mRNA for GH-RH. After PCR products were electrophoresed on agarose gel and stained with ethidium bromide, a 322-bp band, specific for GH-RH, was found in tumor samples as illustrated in Fig. 3ACitation . A band of similar molecular size was also obtained in H-69 SCLC (positive control; Ref. 19 ).



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 3. A, RT-PCR analysis of the expression of mRNA for GH-RH in H838 human NSCLC tumors grown in nude mice. A product of the expected size of 322 bp corresponding to GH-RH was found in mRNA extracts pooled from three to four H838 control tumors (Lanes 1 and 2). Lane M, 100-bp DNA molecular weight marker; Lane P, H-69 human SCLC cell line used as positive control; Lane N, negative control. B, RT-PCR analysis of the expression of mRNA for SVs of GH-RH receptors in H838 human NSCLC tumors grown in nude mice. A product of the expected size of 720 bp corresponding to SV1 was found in mRNA extracts pooled from three to four H838 control tumors (Lanes 1 and 2). Lane N, negative control; Lane M, 100-bp DNA molecular weight marker.

 
RT-PCR Analysis of SV of GH-RH Receptors in H838 NSCLC Tumors.
The expression of mRNA for GH-RH receptors in H838 NSCLC tumors was evaluated by RT-PCR. Using gene-specific primers for amplifying cDNAs for splice variants (SV) of GH-RH receptors (33) , we detected a PCR product of 720 bp in H838 tumors (Fig. 3B)Citation . This PCR product corresponded to SV1 of GH-RH receptors described previously (33) . No bands representing SV2, SV3, and SV4 were obtained in H838 NSCLC tumors.

Radioligand Binding Studies.
The characteristics of specific binding of 125I-labeled GH-RH antagonist JV-1–42 to the membrane receptors on H838 tumor cells were determined using ligand competition assays. The computerized nonlinear curve fitting and the Scatchard plot analyses of the binding data indicated that in membranes of untreated H838 tumors, radiolabeled JV-1–42 was bound to one class of binding sites with a mean dissociation constant (Kd) of 0.84 ± 0.1 nM and a mean maximal binding capacity (Bmax) of 197.5 ± 9.15 fmol/mg membrane protein (Fig. 4)Citation .



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 4. Representative example of saturation of 125I-JV-1–42 binding to H838 NSCLC tumor membrane fraction. The total ({circ}), nonspecific ({blacktriangledown}), and specific ({bullet}) binding is plotted as a function of radiolabeled peptide concentration. Inset, representative Scatchard plot derived from specific 125I-JV-1–42 binding to the membrane fraction isolated from H838 human NSCLC. Each point represents the mean of triplicate determinations.

 
Effect of GH-RH, GH-RH Antagonist JV-1–38, an Antiserum to GH-RH, and IGF-I on the Proliferation of H838 NSCLC Cells in Vitro.
H838 NSCLC cells cultured in vitro were exposed to various concentrations of GH-RH1–29NH2, GH-RH antagonist JV-1–38, GH-RH antiserum, or IGF-I, and the effect on the proliferation was followed by crystal violet assay. GH-RH1–29NH2 at 10-7–10-5 M did not affect the growth of H838 cells (data not shown). GH-RH antagonist JV-1–38 at 10-7–10-5 M inhibited the proliferation of cells by 19–52% (P < 0.05–0.001, respectively) as compared with controls (Fig. 5A)Citation . An antiserum to GH-RH at 1:2500 and 1:1000 dilution inhibited the proliferation of H838 cells by 32.1 and 38.8%, respectively (P < 0.001; Fig. 5ACitation ). Exogenous IGF-I, at the concentration of 5–25 ng/ml, stimulated the growth of cells by 19–30% (P < 0.05–0.001; Fig. 5ACitation ).



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 5. A, effect of GH-RH antagonist JV-1–38, an antiserum to GH-RH or IGF-I on the proliferation of H838 NSCLC cell line. Relative cell number in treated, and control plates was determined by crystal violet staining and expressed as percent T/C values, where T = absorbance of treated cultures and C = absorbance of control cultures. Measured absorbance is proportionate to cell number. B, [3H]thymidine incorporation into H838 NSCLC cells. Confluent cell cultures were incubated with 0.1–10 µM GH-RH antagonist JV-1–38, SV-95 antiserum 1:2500 and 1:1000, or 5–25 ng/ml IGF-I. After 20 h of incubation, the cells were pulsed with 0.25 µCi/well [3H]thymidine for 3–4 h. Columns, mean value of six replicates of a representative experiment. Bars, SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
These findings were confirmed by [3H]thymidine incorporation assay into quiescent H838 NSCLC cells. Both the GH-RH antagonist JV-1–38 and the antiserum to GH-RH induced a significant (P < 0.05) inhibition of DNA synthesis by ~50% (Fig. 5B)Citation . IGF-I stimulated [3H]thymidine incorporation only at the highest dose of 25 ng/ml.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although some progress has been made in the past 10 years in the management of patients with NSCLC, the 5-year relative survival rates have not improved substantially (39) . The outlook for patients with NSCLC remains poor (40) and the need for the development of novel therapeutic modalities persists. This study demonstrates that GH-RH antagonist JV-1–38 significantly inhibits the growth of H838 human NSCLC tumor xenografts in nude mice. After 4 weeks of treatment, the tumor volume and the tumor weight of the animals given JV-1–38 were significantly reduced by >50% as compared with those of the controls, and the tumor-doubling time was also significantly extended.

GH-RH antagonists were initially developed for treatment of cancers dependent on hepatic endocrine IGF-I, stimulated by pituitary GH (17 , 20) . However, more recent evidence points toward a major role of local GH-RH and its tumoral receptors in the pathogenesis of various cancers, indicating that most of the antitumor effects of GH-RH antagonists could be exerted through a direct action on the cancer cells (9 , 19 , 27, 28, 29 , 31 , 33, 34, 35 , 47) . It has been established that locally produced GH-RH is present in appreciable quantities both in the normal lung (41) and in NSCLC and other lung tumor specimens (41, 42, 43, 44) . However, the role of GH-RH in NSCLC has not been investigated thus far. We detected mRNA for GH-RH in H838 tumors grown in nude mice. The quantitation of GH-RH secreted by the cells in vitro revealed that its concentration in the culture medium increased between 24 and 48 h of culture. In cell cultures in vitro, GH-RH did not affect the growth of H838 cell line, but both GH-RH antagonist JV-1–38 and an antibody to GH-RH inhibited the proliferation based on crystal violet assay. Similar effects were observed using a quantitative approach such as [3H]thymidine incorporation assay. The lack of stimulation by exogenous GH-RH in vitro might reflect the fact that H838 cells may already produce GH-RH at levels that can cause maximal stimulation of this cell line in vitro. The inhibition of cell proliferation observed in the presence of a GH-RH antiserum supports this hypothesis. The suppression of growth produced by JV-1–38 in vitro is likely due to the ability of this antagonist to block the actions of autocrine GH-RH on the tumoral GH-RH receptors.

Attempts to detect the presence of pituitary-type GH-RH receptors in cancer cells and tumors were unsuccessful (14 , 26 , 33 , 45 , 46) . However, recently we were able to identify splice variants of GH-RH receptors on tumors that might mediate the direct effects of GH-RH and its antagonists (33 , 34) . These tumoral GH-RH receptors are distinct from the pituitary-type GH-RH receptors or receptors for other peptides of the secretin/glucagon family. The isolation and sequencing of cDNAs corresponding to the tumoral GH-RH receptor mRNAs revealed that they are splice variants of the pituitary GH-RH receptors (33) . All of the SVs of the GH-RH receptor have a retained intronic sequence at their 5' end but lack the first three exons (33 , 34) . We assume that the lack of the first three exons observed in SV1 could result in the formation of a tumoral GH-RH receptor protein in which most of the large NH2-terminal extracellular domain, characteristic of the pituitary receptor, is truncated (33 , 34) . Thus, SV1 could encode a functional receptor protein with binding affinity for GH-RH and its analogues different from that of the pituitary GH-RH receptor (34) . The participation of SV1 in cell proliferation signaling has been shown by transfection in NIH-3T3 mouse fibroblast cells (47) . In that study, transfection of 3T3 cells with SV1 produced specific high-affinity binding sites for radiolabeled GH-RH antagonist and conferred a greatly increased sensitivity to GH-RH analogues in the cell proliferation assays. Thus, the expression of SV1 augmented the mitogenic responses of 3T3 cells to GH-RH1–29NH2 or GH-RH agonist JI-38 and the antimitogenic responses to GH-RH antagonist JV-1–38 compared with control cells not expressing SV1 (47) . In addition, using an endometrial carcinoma cell line that normally expresses SV1 receptors, our group demonstrated that ablation of SV1 by an antisense RNA-based approach reduced the rate of cell proliferation in the absence of exogenous GH-RH and decreased the sensitivity of the cells to exogenous GH-RH (48) .

In our investigation, using 125I-labeled GH-RH antagonist JV-1–42 as a new radioligand, we were able to detect tumoral binding sites for GH-RH with a high affinity and specificity in H838 NSCLC tumor tissue. Moreover, RT-PCR analysis revealed the expression of SV1 of GH-RH receptors in the tumors. Because SV1 may participate in the regulation of cell proliferation and mediate the actions of GH-RH and its antagonists (47 , 48) , the direct inhibitory effect of GH-RH antagonist JV-1–38 on the growth of H838 tumors could have been exerted through these receptors. SV1 is also expressed in other normal and malignant human extrapituitary cells and tissues (23 , 27, 28, 29 , 31 , 33 , 34 , 46) .

GH-RH belongs to the family of structurally related peptide hormones that also includes secretin, glucagon, vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide as well as their receptors are present in NSCLC and may be involved in the autocrine regulation of this malignancy (49, 50, 51, 52) . GH-RH appears to fulfill some of the parameters for an autocrine growth factor in H838 human NSCLC cell line, because it is apparently expressed and secreted by the tumor and a tumoral GH-RH receptor isoform is also present on tumors. GH-RH antagonists can inhibit H838 tumor growth in vivo presumably by an action on GH-RH receptors nullifying the effect of locally produced GH-RH.

Various studies demonstrate the involvement of IGF-I, IGF-II, and IGF-IR in proliferation of NSCLC (21 , 22 , 52 , 53) . Thus, Quinn et al. (22) found that IGF-I and IGF-II mRNAs were coexpressed with IGF-IR in the majority of human lung cancer cell lines. In our study, we have also found that H838 tumors express the mRNA for IGF-IR, which is known to mediate the mitogenic effects of IGF-I (53) . Zia et al. (21) reported that monoclonal antibody to IGF-I receptor inhibited growth of NSCLC in vitro and in vivo. Recently, a statistically significant positive association was reported between plasma IGF-I levels and the risk of lung cancer (54) . Immunoreactive IGF-I is detectable in primary lung tumor tissue including non-SCLC (squamous cell and adenocarcinomas) at higher levels than in normal lung (53) . Immunoreactive IGF-I is secreted into the media of both small cell lung cancer (SCLC) and non small cell lung cancer (NSCLC) cells grown in culture (53) . In our oncological study in vivo, JV-1–38 was administered at a low dose that had only a weak effect on the pituitary GH-hepatic IGF-I axis, as seen from the lack of a significant reduction of serum IGF-I level. Nevertheless, significant decreases in the tumoral concentration of IGF-I and its mRNA expression were observed after treatment with the GH-RH antagonist. Thus, the inhibition of synthesis of IGF-I in H838 tumor tissue could be a consequence of the direct effect of the GH-RH antagonist on the H838 cells in vivo, mediated by the tumoral GH-RH receptors, although the same effect could not be reproduced in vitro during a shorter, 48-h treatment period. This phenomenon would be similar to that observed in IGF-II-producing prostatic, pancreatic, colorectal and ovarian cancers, glioblastomas and sarcomas in vivo, where the treatment with GH-RH antagonists caused an inhibition of the synthesis of tumoral IGF-II (10, 11, 12, 13 , 16 , 18 , 45 , 46) . The ability of the GH-RH antagonist to reduce the concentration of IGF-I in H838 tumor is likely to provide some contribution to the antitumor effect, because IGF-I is a growth factor for this NSCLC. In our experiments in vitro, exogenous IGF-I significantly stimulated the proliferation of H838 cells. However, the inhibition of tumoral IGF-I synthesis could be of relatively minor importance, because in vitro the GH-RH antagonist was able to decrease the proliferation of H838 cells without exerting a parallel inhibitory effect on the synthesis of autocrine IGF-I. Thus, the antiproliferative effect of blocking of GH-RH receptors in vitro may not be mechanistically linked to an inhibition of IGF-I signaling through the suppression of IGF-I synthesis/secretion. In addition, the decreases in tumoral IGF-I protein concentrations appear to be smaller than those in the mRNA levels, although no firm conclusions could be drawn, because only four control tumors and two treated tumors were examined for protein content, because of a shortage of tumor tissue samples.

In conclusion, our work extends previous findings suggesting an autocrine role for GH-RH in various cancers and demonstrates that GH-RH antagonists inhibit the growth of H838 human NSCLC tumors in nude mice, probably by interfering with the stimulatory effects of tumoral (autocrine) GH-RH. Inhibitory effects on the autocrine and endocrine IGF-I system could be also involved in the antitumor mechanisms of GH-RH antagonists, but to a lesser extent. Our findings indicate the merit of further investigations with GH-RH antagonists in the management of NSCLC. Our studies suggest that future therapies, based on GH-RH antagonists, might be beneficial for at least a subset of patients with non small cell lung carcinomas that express GH-RH and IGF-I receptors and are dependent on autocrine stimulation by GH-RH and/or IGF-I.


   ACKNOWLEDGMENTS
 
We thank Brady Wagner and Elena Glotser for their excellent experimental assistance.


   FOOTNOTES
 
Grant support: Medical Research Service of the Veterans Affairs Department and a grant from Zentaris AG (Frankfurt am Main, Germany) to Tulane University (all to A. V. S.).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Andrew V. Schally, M.D., Endocrine, Polypeptide and Cancer Institute, VA Medical Center, 1601 Perdido Street, New Orleans, Louisiana 70112-1262. Phone: (504) 589-5230; Fax: (504) 566-1625.

1 The abbreviations used are: NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer; GH-RH, growth hormone-releasing hormone; IGF-I, insulin-like growth factor I; SV, splice variant; NOR, nucleolar organizer region; SFM, serum-free medium; N2E medium, DMEM with 2 mM L-glutamine, 1 µg/ml transferrin, 30 nM selenium, 20 nM progesterone, and 100 µM putrescine; IGF-IR, IGF-I receptor. Back

Received 5/ 6/03. Revised 7/ 3/03. Accepted 9/ 4/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Levi F., Lucchini F., Negri E., Boyle P., LaVecchia C. Cancer mortality in Europe, 1990–1994, and an overview of trends from 1955 to 1994. Eur. J. Cancer, 35: 1477-1516, 1999.
  2. Greenlee R. T., Hill-Harmon M. B., Murray T., Thun M. Cancer Statistics, 2001. CA Cancer J. Clin., 51: 15-36, 2001.[Abstract/Free Full Text]
  3. Parkin D. M., Pisani P., Ferlay J. Global cancer statistics. CA Cancer J. Clin., 49: 33-64, 1999.[Abstract/Free Full Text]
  4. Schiller J. H. Current standards of care in small-cell and non-small-cell lung cancer. Oncology, 61 (Suppl. 1): 3-13, 2001.
  5. Ginsberg R. J., Vokes E. E., Rosenzweig K. Non-small cell lung cancer DeVita V. T., Jr. Hellman S. Rosenberg S. A. eds. . Cancer: Principles and Practice of Oncology, 925-951, Lippincott Williams & Wilkins Philadelphia 2001.
  6. Scagliotti G. V., Novello S., Selvaggi G. Multidrug resistance in non-small-cell lung cancer. Ann. Oncol., 10 (Suppl. 50): 83-86, 1979.
  7. Varga J. L., Schally A. V., Csernus V. J., Zarandi M., Halmos G., Groot K., Rekasi Z. Synthesis and biological evaluation of antagonists of growth hormone-releasing hormone with high and protracted in vivo activities. Proc. Natl. Acad. Sci. USA, 96: 692-697, 1999.[Abstract/Free Full Text]
  8. Schally A. V., Varga J. L. Antagonistic analogs of growth hormone-releasing hormone (GH-RH): new potential antitumor agents. Trends Endocrinol. Metab., 10: 383-391, 1999.[Medline]
  9. Schally A. V., Comaru-Schally A. M., Nagy A., Kovacs M., Szepeshazi K., Plonowski A., Varga J. L., Halmos G. Hypothalamic hormones and cancer. Front. Neuroendocrinol., 22: 248-291, 2001.[Medline]
  10. Szepeshazi K., Schally A. V., Groot K., Armatis P., Hebert F., Halmos G. Antagonists of growth hormone releasing hormone (GH-RH) inhibit in vivo proliferation of experimental pancreatic cancers and decrease IGF-II levels in tumors. Eur. J. Cancer, 36: 128-136, 2000.
  11. Szepeshazi K., Schally A. V., Groot K., Armatis P., Halmos G., Hebert F., Szende B., Varga J. L., Zarandi M. Antagonists of growth hormone-releasing hormone (GH-RH) inhibit IGF-II production and growth of HT-29 human colon cancers. Br. J. Cancer, 82: 1724-1731, 2000.[Medline]
  12. Jungwirth A., Schally A. V., Pinski J., Halmos G., Groot K., Armatis P., Vadillo-Buenfil M. Inhibition of in vivo proliferation of androgen-independent prostate cancers by an antagonist of growth hormone-releasing hormone. Br. J. Cancer, 75: 1585-1592, 1997.[Medline]
  13. Lamharzi N., Schally A. V., Koppan M., Groot K. Growth hormone-releasing hormone antagonist MZ-5–156 inhibits growth of DU-145 human androgen-independent prostate carcinoma in nude mice and suppresses the levels and mRNA expression of insulin-like growth factor II in tumors. Proc. Natl. Acad. Sci. USA, 95: 8864-8868, 1998.[Abstract/Free Full Text]
  14. Kahan Z., Varga J. L., Schally A. V., Rekasi Z., Armatis P., Chatzistamou I., Czompoly T., Halmos G. Antagonists of growth hormone releasing hormone arrest the growth of MDA-MB-468 estrogen-independent human breast cancers in nude mice. Breast Cancer Res. Treatment, 60: 71-79, 2000.[Medline]
  15. Jungwirth A., Schally A. V., Pinski J., Groot K., Armatis P., Halmos G. Growth hormone-releasing hormone antagonist MZ-4–71 inhibits in vivo proliferation of CAKI-1 renal adenocarcinoma. Proc. Natl. Acad. Sci. USA, 94: 5810-5813, 1997.[Abstract/Free Full Text]
  16. Kiaris H., Schally A. V., Varga J. L. Antagonists of growth hormone-releasing hormone inhibit the growth of U-87MG human glioblastoma in nude mice. Neoplasia, 2: 242-250, 2000.[Medline]
  17. Pinski J., Schally A. V., Groot K., Halmos G., Szepeshazi K., Zarandi M., Armatis P. Inhibition of growth of human osteosarcomas by antagonists of growth hormone-releasing hormone. J. Natl. Cancer Inst., 87: 1787-1794, 1995.[Abstract/Free Full Text]
  18. Braczkowski R., Schally A. V., Plonowski A., Varga J. L., Groot K., Krupa M., Armatis P. Inhibition of proliferation in human MNNG/HOS osteosarcoma and SK-ES-1 Ewing’s sarcoma cell line in vitro and in vivo by antagonists of growth hormone-releasing hormone: effects on IGF-II. Cancer (Phila.), 95: 1735-1745, 2002.
  19. Kiaris H., Schally A. V., Varga J. L., Groot K., Armatis P. Growth hormone-releasing hormone: an autocrine growth factor for small cell lung carcinoma. Proc. Natl. Acad. Sci. USA, 96: 14894-14898, 1999.[Abstract/Free Full Text]
  20. Pinski J., Schally A. V., Jungwirth A., Groot K., Halmos G., Armatis P., Zarandi M., Vadillo-Buenfil M. Inhibition of growth of small cell and non-small cell lung carcinomas by antagonist of growth hormone-releasing hormone (GH-RH). Int. J. Oncol., 9: 1099-1105, 1996.
  21. Zia F., Jacobs S., Kull F., Jr., Cuttitta F., Mulshine J. L., Moody T. W. Monoclonal antibody {alpha}IR-3 inhibits non-small cell lung cancer growth in vitro and in vivo. J. Cell. Biochem. Suppl., 24: 269-275, 1996.[Medline]
  22. Quinn K. A., Treston A. M., Unsworth E. J., Miller M. J., Vos M., Grimley C., Battey J., Mulshine J. L., Cuttitta F. Insulin-like growth factor expression in human cancer cell lines. J. Biol. Chem., 19: 11477-11483, 1996.
  23. Chatzistamou I., Schally A. V., Varga J. L., Groot K., Busto R., Armatis P., Halmos G. Inhibition of growth and metastases of MDA-MB-435 human estrogen-independent breast cancers by an antagonist of growth hormone-releasing hormone. Anticancer Drugs, 12: 761-768, 2001.[Medline]
  24. Szepeshazi K., Schally A. V., Armatis P., Groot K., Hebert F., Feil A., Varga J. L., Halmos G. Antagonists of GH-RH decrease production of GH and IGF-I in MXT mouse mammary cancers and inhibit tumor growth. Endocrinology, 142: 4371-4378, 2001.[Abstract/Free Full Text]
  25. Csernus V. J., Schally A. V., Kiaris H., Armatis P. Inhibition of growth, production of insulin-like growth factor-II (IGF-II) and expression of IGF-II mRNA of human cancer cell-lines by antagonistic analogs of growth hormone-releasing hormone in vitro. Proc. Natl. Acad. Sci. USA, 96: 3098-3103, 1999.[Abstract/Free Full Text]
  26. Rekasi Z., Varga J. L., Schally A. V., Halmos G., Armatis P., Groot K., Czompoly T. Antagonists of growth hormone-releasing hormone and vasoactive intestinal peptide inhibit tumor proliferation by different mechanisms: evidence from in vitro studies on human prostatic and pancreatic cancers. Endocrinology, 141: 2120-2128, 2000.[Abstract/Free Full Text]
  27. Busto R., Schally A. V., Braczkowski R., Plonowski A., Krupa M., Groot K., Armatis P., Varga J. L. Expression of mRNA for growth hormone-releasing hormone (GH-RH), and splice variants of GH-RH receptors in human malignant bone tumors. Regul. Pept., 108: 47-53, 2002.[Medline]
  28. Chopin L. K., Herington A. C. A potential autocrine pathway for growth hormone releasing hormone (GH-RH) and its receptor in human prostate cancer cell lines. Prostate, 49: 116-121, 2001.[Medline]
  29. Plonowski A., Schally A. V., Busto R., Krupa M., Varga J. L., Halmos G. Expression of growth hormone-releasing hormone (GH-RH) and splice variants of GH-RH receptors in human experimental prostate cancers. Peptides, 23: 1127-1133, 2002.[Medline]
  30. Kahan Z., Arencibia J. M., Csernus V. J., Groot K., Kineman R. D., Robinson W. R., Schally A. V. Expression of growth hormone-releasing hormone (GH-RH) messenger ribonucleic acid and the presence of biologically active GH-RH in human breast, endometrial, and ovarian cancers. J. Clin. Endocrinol. Metab., 84: 582-589, 1999.[Abstract/Free Full Text]
  31. Halmos G., Schally A. V., Czompoly T., Krupa M., Varga J. L., Rekasi Z. Expression of growth hormone-releasing hormone (GH-RH) and its receptor splice variants in human prostate cancer. J. Clin. Endocrinol. Metab., 87: 4707-4714, 2002.[Abstract/Free Full Text]
  32. Benlot C., Levy L., Fontanaud P., Roche A., Rouannet P., Joubert D. Somatostatin and growth hormone-releasing hormone in normal and tumoral human breast tissue: endogenous content, in vitro pulsatile release, and regulation. J. Clin. Endocrinol. Metab., 82: 690-696, 1997.[Abstract/Free Full Text]
  33. Rekasi Z., Czompoly T., Schally A. V., Halmos G. Isolation and sequencing of cDNA for splice variants of growth hormone releasing hormone receptors from human cancers. Proc. Natl. Acad. Sci. USA, 97: 10561-10566, 2000.[Abstract/Free Full Text]
  34. Halmos G., Schally A. V., Varga J. L., Plonowski A., Rekasi Z., Czompoly T. Human renal cell carcinoma expresses distinct binding sites for growth hormone-releasing hormone. Proc. Natl. Acad. Sci. USA, 97: 10555-10560, 2000.[Abstract/Free Full Text]
  35. Kineman R. D. Antitumorigenic actions of growth hormone-releasing hormone antagonists. Proc. Natl. Acad. Sci. USA, 97: 532-534, 2000.[Free Full Text]
  36. Smolev J. K., Heston W. D. W., Scott W. W., Coffey D. S. Characterization of the Dunning R3327H prostatic adenocarcinoma: an appropriate animal model for prostate cancer. Cancer Treat. Rep., 61: 273-287, 1997.
  37. Bernhardt G., Reile H., Birnbock H., Spruss T., Schoenenberger H. Standardized kinetic microassay to quantify differential chemosensitivity on the basis of proliferative activity. J. Cancer Res. Clin. Oncol., 118: 35-43, 1992.[Medline]
  38. Groot K., Csernus V. J., Pinski J., Zsigo J., Rekasi Z., Zarandi M., Schally A. V. Development of a radioimmunoassay for some agonists of growth hormone-releasing hormone. Int. J. Pept. Prot. Res., 41: 162-168, 1993.
  39. Travis W. D., Travis L. B., Devesa S. S. Lung cancer. Cancer, 75: 191-202, 1995.[Medline]
  40. Minna J. D. Neoplasms of the lung Braunwald E. Isselbacker K. J. Petersdorf R. G. eds. . Harrison’s Principles of Internal Medicine, Ed. 11 1115-1125, McGraw Hill New York 1987.
  41. Losa M., Wolfram G., Mojto J., Schopohl J., Spiess Y., Huber R., Muller O. A., von Werder K. Presence of growth hormone-releasing hormone-like immunoreactivity in human tumors: characterization of immunological and biological properties. J. Clin. Endocrinol. Metab., 70: 62-68, 1990.[Abstract]
  42. Nomori H., Kobayashi R., Kubo A., Morinaga S., Shintani Y., Sano T. Lung cancer containing growth hormone-releasing hormone associated with hypertrophic osteoarthropathy. Case report. Scand. J. Thorac. Cardiovasc. Surg., 28: 149-152, 1994.[Medline]
  43. Southgate J. H., Archbold P. G., el-Sayed E. M., Wright J., Marks V. Ectopic release of GH-RH and ACTH from an adenoid cystic carcinoma resulting in acromegaly and complicated by pituitary infarction. Postgrad. Med. J., 64: 145-148, 1988.[Abstract]
  44. Asa S. L., Kovacs K., Thorner M. O., Leong D. A., Rivier J., Vale W. Immunological localization of growth hormone-releasing hormone in human tumors. J. Clin. Endocrinol. Metab., 60: 423-427, 1985.[Abstract]
  45. Plonowski A., Schally A. V., Varga J. L., Rekasi Z., Hebert F., Halmos G., Groot K. Potentiation of the inhibitory effect of growth hormone-releasing hormone antagonists on PC-3 human prostate cancer by bombesin antagonists indicative of interference with both IGF and EGF pathways. Prostate, 44: 172-180, 2000.[Medline]
  46. Chatzistamou I., Schally A. V., Varga J. L., Groot K., Armatis P., Busto R., Halmos G. Antagonists of growth hormone-releasing hormone and somatostatin analog RC-160 inhibit the growth of OV-1063 human epithelial ovarian cancer cell line xenografted into nude mice. J. Clin. Endocrinol. Metab., 86: 2144-2152, 2001.[Abstract/Free Full Text]
  47. Kiaris H., Schally A. V., Busto R., Halmos G., Artavanis-Tsakonas S., Varga J. L. Expression of a splice variant of the receptor for GH-RH in 3T3 fibroblasts activates cell proliferation responses to GH-RH analogs. Proc. Natl. Acad. Sci. USA, 99: 196-200, 2002.[Abstract/Free Full Text]
  48. Kiaris H., Chatzistamou I., Schally A. V., Halmos G., Varga J. L., Koutselini H., Kalofoutis A. Ligand-dependent and -independent effects of splice variant 1 of growth hormone-releasing hormone receptor. Proc. Natl. Acad. Sci. USA, 100: 9512-9517, 2003.[Abstract/Free Full Text]
  49. Reubi J. C., Laderach U., Waser B., Gebbers J. O., Robberecht P., Laissue J. A. Vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor subtype in human tumors and their tissues of origin. Cancer Res., 60: 3105-3112, 2000.[Abstract/Free Full Text]
  50. Zia F., Fagarasan M., Kamil B., Coy D. H., Pisegna J. R., Wank S. A., Moody T. W. Pituitary adenylate cyclase activating peptide receptors regulate the growth of non-small cell lung cancer cells. Cancer Res., 55: 4886-4891, 1995.[Abstract/Free Full Text]
  51. Moody T. W., Zia F., Draoui M., Brenneman D. E., Fridkin M. A vasoactive intestinal peptide antagonist inhibits non-small lung cancer growth. Proc. Natl. Acad. Sci. USA, 90: 4345-4349, 1993.[Abstract/Free Full Text]
  52. Moody T. W. Peptides and growth factors in non-small cell lung cancer. Peptides, 17: 545-555, 1996.[Medline]
  53. Macaulay V. M. Insulin-like growth factors and cancer. Br. J. Cancer, 65: 311-320, 1992.[Medline]
  54. Yu H., Spitz M. R., Mistry J., Gu J., Hong W. K., Wu X. Plasma levels of insulin-like growth factor-I and lung cancer risk: a case-control analysis. J. Natl. Cancer Inst., 91: 151-156, 1999.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Buchholz, A. V. Schally, J. B. Engel, F. Hohla, E. Heinrich, F. Koester, J. L. Varga, and G. Halmos
Potentiation of mammary cancer inhibition by combination of antagonists of growth hormone-releasing hormone with docetaxel
PNAS, February 6, 2007; 104(6): 1943 - 1946.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
D. Sachdev and D. Yee
Disrupting insulin-like growth factor signaling as a potential cancer therapy
Mol. Cancer Ther., January 1, 2007; 6(1): 1 - 12.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
B. S. Miller and D. Yee
Type I Insulin-like Growth Factor Receptor as a Therapeutic Target in Cancer
Cancer Res., November 15, 2005; 65(22): 10123 - 10127.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
G. Keller, A. V. Schally, K. Groot, G. L. Toller, A. Havt, F. Koster, P. Armatis, G. Halmos, M. Zarandi, J. L. Varga, et al.
Effective treatment of experimental human non-Hodgkin's lymphomas with antagonists of growth hormone-releasing hormone
PNAS, July 26, 2005; 102(30): 10628 - 10633.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
J. B. Engel, G. Keller, A. V. Schally, G. L. Toller, K. Groot, A. Havt, P. Armatis, M. Zarandi, J. L. Varga, and G. Halmos
Inhibition of Growth of Experimental Human Endometrial Cancer by an Antagonist of Growth Hormone-Releasing Hormone
J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3614 - 3621.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
S. K. Frankel, B. M. Moats-Staats, C. D. Cool, M. W. Wynes, A. D. Stiles, and D. W. H. Riches
Human insulin-like growth factor-IA expression in transgenic mice promotes adenomatous hyperplasia but not pulmonary fibrosis
Am J Physiol Lung Cell Mol Physiol, May 1, 2005; 288(5): L805 - L812.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar