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Endocrinology Vol. 143, No. 2 484-491
Copyright © 2002 by The Endocrine Society

CANCER

The Antiproliferative Effect of Synthetic Peptidyl GH Secretagogues in Human CALU-1 Lung Carcinoma Cells

Corrado Ghè, Paola Cassoni, Filomena Catapano, Tiziana Marrocco, Romano Deghenghi, Ezio Ghigo, Giampiero Muccioli and Mauro Papotti

Departments of Anatomy, Pharmacology and Forensic Medicine (C.G., F.C., G.M.), Biomedical Sciences and Oncology (P.C., T.M., M.P.), Internal Medicine (E.G.), University of Turin, Turin 10125, Italy; and Europeptides (R.D.), Argenteuil, France

Address all correspondence and requests for reprints to: Giampiero Muccioli, Ph.D., Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin, Via P. Giuria 13, 10125 Turin, Italy. E-mail: giampiero.muccioli{at}unito.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The specific binding of [125I]Tyr-Ala-hexarelin, a radiolabeled peptidyl GH secretagogue (GHS), has been investigated in nontumoral and neoplastic human lung tissues. This binding was very marked in nonendocrine lung carcinomas with values that were greater than found in either normal lung or in endocrine lung neoplasms. Tyr-Ala-hexarelin binding was also present in a human lung carcinoma cell line (CALU-1). [125I]Tyr-Ala-hexarelin binding to tumor membranes was displaced by peptidyl GHS (GHRP-6, hexarelin) and EP-80317, an hexarelin analog devoid of GH-releasing activity in vivo. In contrast, no competition was observed in the presence of the nonpeptidyl GHS MK-0677 and the endogenous ligand of the GHS-R1a ghrelin. GHS-R1a mRNA expression was found in 50% of endocrine lung tumors but was never seen in other nontumoral and neoplastic lung tissues nor in CALU-1. In these cells, hexarelin and EP-80317, but not ghrelin or MK-0677, caused a dose-dependent inhibition of IGF-II-stimulated thymidine incorporation and cell growth at concentrations close to their binding affinity. In conclusion, this study shows that inhibition of DNA synthesis and proliferation of CALU-1 cells is caused by peptidyl but not by nonpeptidyl GHS and ghrelin and suggests that this effect is likely to be mediated by a specific non-GHS-R1a receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH SECRETAGOGUES (GHS) are synthetic peptidyl and nonpeptidyl molecules that possess strong, dose-dependent and reproducible GH-releasing activity in vivo but also significant PRL- and ACTH/cortisol-releasing effects (1, 2). The neuroendocrine activities of GHS are mediated by a G protein-coupled receptor, the GHS receptor type 1a (GHS-R1a), which has originally been identified in the pituitary and the hypothalamus in humans, as well as in rats, using a radiolabeled nonpeptidyl ([35S]MK-0677) GHS (3, 4, 5). Recently, an endogenous ligand has been described for this receptor and named ghrelin (6, 7). This is a gastric-derived peptide that binds with high affinity to the hypothalamic and pituitary GHS-R1a (8, 9) and, in rat and in man, possesses stimulatory effects on GH, ACTH, and PRL secretion similar to those of synthetic GHS (10, 11, 12). Ghrelin and all the GHS compounds developed so far seem to exhibit a high binding affinity to the cloned GHS-R1a. (3, 8, 9, 13). However, there is strong evidence suggesting the existence of additional receptor subtypes that may exhibit different affinities for these compounds. In fact, sites with high affinity for Tyr-Ala-hexarelin and other peptidyl GHS have been found in rat and human heart (14, 15, 16), as well as in a wide range of other nonendocrine peripheral tissues (17, 18). These binding sites are presumably non-GHS-R1a because they have a very low binding affinity for ghrelin and MK-0677 (18). The functional significance of peripheral GHS-R is still unknown. GHS-R1a is expressed in the heart and ghrelin shares with peptidyl GHS some (19) but not other cardiotropic activities; and it is possible that these latter activities may be mediated by binding sites which are specific only for peptidyl GHS (20, 21, 22). Moreover, non-type 1a GHS-R have been found in some human neoplasms such as thyroid tumors of follicular origin and breast cancer and probably mediate the inhibitory activity of peptidyl GHS on the growth of these tumors (23, 24).

It has already been shown that the human lung contains a considerable number of non-GHS-R1a (18) but, at present, there is no information about the occurrence of these receptors in lung tumors, as well as on the possibility that GHS may directly influence lung tumor growth. We have therefore investigated in nontumoral lung tissue, in nonendocrine (carcinomas) and endocrine neoplasms (carcinoid tumors) from human lung, as well as in a human lung carcinoma cell line (CALU-1): 1) the specific binding of [125I]Tyr-Ala-hexarelin and the competition with such binding by natural (ghrelin) and synthetic GHS (either classical GHS such as hexarelin, GHRP6, MK-0677, or new peptidyl GHS analogs such as EP-80317 and EP-9399, which are devoid of GH-releasing activity in vivo); 2) the presence of GHS-R1a mRNA expression; and 3) the effects of natural and synthetic GHS on the proliferation of CALU-1 human lung carcinoma cell line in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Human ghrelin-(1–28), MK-0677, GHRP-6, hexarelin and three structurally related analogs of hexarelin, such as Tyr-Ala-hexarelin, EP-80317 [(2S,5S)-5-amino-1,2,3,4,6,7-hexahydro-azepino (3,2,1-hi)indol-4-one-2-carboxylic acid-D-2Me-Trp-D-Lys-Trp-D-Phe-Lys-NH2)] and the cyclic derivative EP-9399 [c(Trp-D-Phe-His-2Me-Trp-Ala)] were provided by Europeptides (Argenteuil, France). Human GHRH-(1–29), SRIF-(1–14) and insulin-like growth factor-II (IGF-II) were purchased from Bachem Feinchemikalien AG (Bubendorf, Switzerland). [125I]Tyr-Ala-hexarelin (specific activity: 2000 Ci/mmol) was iodinated using a lactoperoxidase method and purified by reverse-phase HPLC, as previously described (25, 26). [3H]Thymidine (specific activity 2000 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Milan, Italy). Penicillin, streptomycin, FCS, trypsin/EDTA solution, and other tissue culture reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD).

Cell culture
CALU-1 cell line derived from a human nonendocrine lung carcinoma was purchased from the ATCC (Manassas, VA). Cells were grown in DMEM supplemented with FCS 10% and penicillin/streptomycin (standard culture medium) in a 5% CO2 humidified atmosphere at 37 C and used in binding and cell proliferation studies.

Tissue samples
Several autoptic nontumoral lung samples and surgically resected lung tumors were included in this study. All postmortem lung samples were obtained at autopsy from 10 male subjects ranging in age from 25 to 61 yr (median age 47 yr) who died of trauma and were subjected to autopsy for diagnostic purposes in years 1998–2000 in the Department of Pathology, University of Turin. Twenty lung tumors [12 nonendocrine carcinomas and 8 endocrine neoplasms (carcinoids)] were collected from surgical specimens received in the above Department in the same period. Tumors were classified according to the WHO classification of lung cancer (27). All patients [20 males ranging in age from 39 to 65 yr (median age 55 yr)] gave their informed consent for the research use of their tissues and the study obtained ethical approval by an independent local Ethical Committee. A sample of tissue immediately adjacent to the portion taken for histopathological diagnosis was immediately frozen at -80 C and stored for 4–24 months until processed for membrane preparation and binding studies or for RT-PCR analysis.

Binding studies
[125I]Tyr-Ala-hexarelin binding to membranes (30,000 x g pellet) isolated from CALU-1 cells and nontumoral and neoplastic human tissue was carried out as previously described (18, 25). Tyr-Ala-hexarelin has been reported to have the same GH-releasing potency of hexarelin in rats (26) and humans ( 28) and to be a reliable probe for labeling GHS-R in human tissues (18, 25). In preliminary experiments, the optimal binding conditions for the lung carcinoma cells were found to be similar to those previously reported for other human tissues (18). For single point binding assay, cell or tissue membranes (corresponding to 100 µg of membrane protein, measured using the method of Lowry et al. (29) were incubated in triplicate at 0 C for 1 h with approximately 5 nM [125I]Tyr-Ala-hexarelin in a final volume of 0.5 ml assay buffer (50 mM Tris, 2 mM EGTA, 0.1% BSA, 0.03% bacitracin, titrated with HCl to pH 7.3). Parallel incubations, where 2.5 µM unlabeled Tyr-Ala-hexarelin was also present, were used to determine nonspecific binding that was subtracted from total binding to yield specific binding values. The binding reaction was terminated by adding ice-cold assay buffer followed by filtration through Whatman GF/B filters. Filters were rinsed three times with assay buffer and the radioactivity bound to membranes was measured by a Packard auto-{gamma} counter. Specific binding was expressed as a percentage of the total radioactivity added. Precautions were taken to minimize variations in the binding of [125I]Tyr-Ala-hexarelin to tissue membranes. Thus, all binding studies related to one membrane preparation were carried out using the same batch of radiotracer. In some assays, receptor binding saturation studies were also conducted by incubating tissue membranes with increasing concentrations (from 0.2– 20 nM) of radioligand in the absence and in the presence of a fixed amount (2.5 µM) of unlabeled Tyr-Ala-hexarelin. Saturation isotherms were transformed using the method of Scatchard (30) and the dissociation constant (Kd) and number of binding sites (Bmax) were calculated with the Prism 3 program (GraphPad Software, Inc., San Diego, CA). To establish binding site specificity, increasing concentrations of various competitors were tested in displacement assays with [125I]Tyr-Ala-hexarelin. The concentration of a competitor agent causing 50% inhibition of specific radioligand binding (IC50 value) was derived from the iterative curve-fitting analysis.

RT-PCR for GHS-R1a
Total RNA was extracted from 3 normal lung tissues, 12 nonendocrine carcinomas, and 6 endocrine lung tumors, as well as CALU-1 cells. cDNA transcription was performed as described elsewhere (31). The primer for GHS-R1a was synthesized according to the sequence reported by Korbonits et al. (32) and employed for RT-PCR using the same conditions described by these authors. After RT for 15 min at 60 C, the reaction mixture was denaturated at 92 C for 5 min followed by 40 cycles of PCR. ß2-microglobulin amplification served as a control of the RNA quality (see details in Ref. 31). To further test the RT-PCR product specificity, Southern blot analysis was performed using the probe sequence previously published (32).

Cell proliferation studies
Cell proliferation was evaluated either by [3H]thymidine incorporation into DNA or counting cell number after appropriate incubation with different compounds. [3H]Thymidine incorporation studies were performed as previously described (24). Briefly, starved human CALU-1 lung carcinoma cells (2 x 105 cells/ml) were incubated at 37 C with or without 10 ng/ml IGF-II in the absence or in the presence of different concentrations (from 1 nM to 2 µM) of hexarelin, EP-80317, ghrelin, and MK-0677. After incubation for 20 h, 1 µCi/well of [3H]thymidine was added and the incubation was continued for an additional 4 h. The reaction was then halted and the cells were harvested onto glass-fiber filter strips. Incorporation of [3H]thymidine was measured in a scintillation counter. For cell growth studies, CALU-1 cells were seeded in triplicate in 48-multiwell plates (5 x 103 cells/well) in standard culture medium and allowed to became attached for 24 h. Cells were synchronized, 8 h after plating, by a 36-h rest in 0.5% FCS and grown in standard culture medium for 96 h in the absence or in the presence of 1 µM of the above indicated compounds, with media changed every 48 h. At 48 and 96 h of treatment, respectively, cells were fixed in 2.5% glutaraldehyde, stained with 0.1% crystal violet in 20% methanol, and solubilized in 10% acetic acid. Cell growth was evaluated by measuring absorbance at 590 nm in a microplate reader (Multiskan Bichromatic, Thermo-Labsystems Oy, Helsinki, Finland). A calibration curve was set up with known numbers of cells and a linear correlation between absorbance and cell counts was established up to 1 x 105 cells. In some assays, the effects of different concentrations (ranging from 10 nM to 1 µM) of hexarelin and EP-80317 on cell growth was also studied and the number of cells were determined after 96 h of treatment.

Statistical analysis
Values are expressed as median and range unless otherwise noted. In saturation and competition binding experiments, as well as in cell proliferation studies, data are given as mean ± SEM, unless otherwise specified. The number of cases is indicated by n. Significant differences between groups were assessed by Kruskal-Wallis test. P < 0.05 was chosen as level of significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Binding of [125I]Tyr-Ala-hexarelin to membranes from nontumoral and neoplastic human lung tissue and CALU-1 lung carcinoma cells
Nontumoral lung showed considerable Tyr-Ala-hexarelin specific binding values that were greater than those previously found (11.3–14%) in a classical GHS target tissue, namely the human pituitary gland (25). Tyr-Ala-hexarelin-specific binding was observed in all lung specimens examined and represented about 58–72% of total radioactivity bound. Tyr-Ala-hexarelin binding was very high in nonendocrine lung carcinomas, with values that were 2-fold greater (P < 0.001) than those of the nontumoral lung. In contrast, endocrine lung neoplasms (carcinoid tumors) displayed specific binding values that were close to the binding values recorded in the normal lung. A considerable specific binding was also present in the CALU-1 cell line originated from a nonendocrine human lung carcinoma (Table 1Go).


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  		Table 1. Distribution of [125I]Tyr-Ala-hexarelin binding to membranes of nontumoral and neoplastic human lung tissues and CALU-1 lung cancer cells

 
Saturation studies and specificity of binding
To determine whether the binding of [125I]Tyr-Ala-hexarelin to tissue membranes showed properties typical of the ligand-receptor interaction, the binding of radiotracer was investigated in more detail in the CALU-1 cells and in some specimens of nontumoral and neoplastic lung tissue that yielded sufficient amounts of membranes for these studies. Experiments using increasing concentrations of [125I]Tyr-Ala-hexarelin provided evidence for a saturable specific binding in the nontumoral lung tissue, in a nonendocrine carcinoma, and an endocrine lung carcinoid, as well as in CALU-1 cells (Fig. 1AGo). Scatchard analysis of these data (Fig. 1BGo) demonstrated the existence of a single class of high-affinity sites in all samples examined (Kd values, all expressed as nM, were: 2.2 for nontumoral lung, 2.0 for the carcinoma, 2.1 for the carcinoid tumor, and 1.9 for the CALU-1 cells), with limited binding capacity (Bmax values, all expressed as fmol/mg protein, were: 1,598 for the nontumoral lung, 2,780 for the carcinoma, 1,371 for the carcinoid tumor, and 2,490 for the CALU-1 cells). The calculated Bmax values of Tyr-Ala-hexarelin binding sites in four lung carcinomas were significantly greater (2,934 ± 165 fmol/mg protein, mean ± SEM; P < 0.01) than those measured in lung carcinoid tumors (1,561 ± 119 fmol/mg protein, n = 4) or in the nontumoral lung tissue (1,653 ± 121 fmol/mg protein, n = 4). No difference in the Kd values among the three types of tissues was observed, with values of 2.53 ± 0.4 nM in the nontumoral lung, 2.5 ± 0.5 nM in the carcinomas, and 2.0 ± 0.4 nM in the carcinoid neoplasms.



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  		Figure 1. A and B, Representative saturation of [125I]Tyr-Ala-hexarelin binding to membranes of nontumoral lung, nonendocrine (carcinoma) and endocrine (carcinoid) lung neoplasms, and human lung carcinoma cells (CALU-1 cell line). Experiments were performed by incubating a fixed amount of membrane protein (100 µg/tube) with increasing concentrations of radiolabeled Tyr-Ala-hexarelin alone (total binding) or plus 2.5 µM unlabeled Tyr-Ala-hexarelin to define nonspecific binding. Specific binding values were obtained by subtracting nonspecific binding from total binding (A). The saturation curves of specific binding were analyzed by Scatchard analysis (B) to calculate the maximal binding capacities (Bmax) and the dissociation constants (Kd).

 
The specificity of [125I]Tyr-Ala-hexarelin binding to membranes from lung carcinomas and CALU-1 cells was established by competitive binding experiments using some peptidyl GHS (hexarelin and GHRP6), and two hexarelin derivatives (EP-80317 and the cyclic hexarelin analog EP-9399) that do not have GH-releasing activity in vivo (Ref. 33 and Locatelli V., T. Reissmann, I. C. Robinson, personal communications). A nonpeptidyl GHS such as MK-0677, human ghrelin, an endogenous ligand of the GHS-R type 1a (6), and various hormones that have been reported to stimulate (GHRH, IGF-II) or inhibit (SRIF-14) the growth of endocrine lung cancer cells (34, 35, 36) were also studied in these competitive binding experiments. [125I]Tyr-Ala-hexarelin bound to lung carcinoma membranes (Fig. 2AGo) was completely displaced in a dose-dependent manner by unlabeled hexarelin, GHRP-6, and EP-80317, all of which exhibited high affinity with IC50 values (mean ± SEM of four separate experiments) of 41.0 ± 4.3 nM (hexarelin), 60 ± 7.3 nM (GHRP-6), and 33.5 ± 5.9 nM (EP-80317). In contrast, no competition was observed in the presence of MK-0677, ghrelin, EP-9399, GHRH, SRIF-14, or IGF-II. The pattern of displacement specificities in the CALU-1 cells overlapped that of the tumor tissue (Fig. 2BGo).



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  		Figure 2. A and B, Displacement of [125I]Tyr-Ala-hexarelin from membranes of human nonendocrine lung carcinomas (A) and CALU-1 lung carcinoma cells (B) by different unlabeled competitors. Binding assays were conducted as described in Materials and Methods. The ordinate represents binding as a percentage of control (specific binding in the absence of unlabeled competitor). Values are mean ± SEM of four separate experiments.

 
Expression of GHS-R1a mRNA
By RT-PCR (Fig. 3Go), a 65-bp signal corresponding to GHS-R1a mRNA was observed in the human pituitary used as a positive control and in 3 of 6 endocrine tumors of the lung. No signal was observed in any of the 12 nonendocrine carcinomas tested nor in the CALU-1 lung carcinoma cells. Nontumoral lung was negative, as well.



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  		Figure 3. Expression of GHS-R1a, by RT-PCR, in 6 human lung endocrine tumors (lanes 1–6), 12 nonendocrine lung carcinomas (lanes 7–18), 3 nontumoral lung specimens (lanes 19–21), and in the CALU-1 human lung carcinoma cells (lane 22). Lane 23 represents the signal obtained in the absence of reverse transcriptase enzyme. Human pituitary (lane C) has been used as positive control. W, Water.

 
Effect of hexarelin, EP-80317, MK-0677, and ghrelin on CALU-1 cell proliferation
Based on the evidence of specific [125I]Tyr-Ala-hexarelin binding sites in CALU-1 cells and on the displacement data that have been discussed above, we investigated the effects of hexarelin, EP-80317, MK-0677, and ghrelin on the proliferation of these cells in vitro. Both the basal [3H]thymidine incorporation (serum-free conditions) and that stimulated by IGF-II were studied on the CALU-1 cells. Figure 4Go shows that hexarelin and EP-80317 inhibited the IGF-II-stimulated [3H]thymidine incorporation in a concentration-dependent manner. The calculated 50% effective doses (mean ± SEM of four separate experiments) were 60.3 ± 5.7 nM for hexarelin and 49.0 ± 5.7 nM for EP-80317. In contrast, no inhibition was observed in the presence of ghrelin or MK-0677, which were unable to displace [125I]Tyr-Ala-hexarelin binding to membranes from CALU-1 cells. In addition, no change in thymidine incorporation was observed when the different compounds were incubated with CALU-1 cells growing in serum-free conditions.



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  		Figure 4. Effect of hexarelin, EP-80317, ghrelin, and MK-0677 on basal (dashed line) and IGF-II-stimulated (solid line) incorporation of [3H]thymidine into DNA by human CALU-1 lung carcinoma cells. DNA synthesis was estimated by incorporation of [3H]thymidine after a 20-h incubation with or without 10 ng/ml IGF-II in the absence or in the presence of different concentrations of the indicated compounds. Data are the average of duplicate assay determinants and similar results were obtained in at least two other independent measurements.

 
Experiments on cell growth revealed that hexarelin and EP-80317, but not ghrelin and MK-0677, induced a significant inhibition of cell proliferation in CALU-1 cells, at a concentration (1 µM) that has been found to fully displace [125I]Tyr-Ala-hexarelin from binding sites. At 48 h after starting treatment, hexarelin and EP-80317 determined a significant decrease in cell number (ranging from 30 to 60%), compared with controls, and at 96 h of treatment the decrease of cell number was even more evident and ranged from 59 to 68% inhibition (Fig. 5Go). In addition, both hexarelin and EP-80317 caused a dose-dependent inhibition of cell proliferation at concentrations quite close to their binding affinity (Fig. 6Go). The calculated 50% effective doses (mean ± SEM of three separate experiments) were 64 ± 14 nM for hexarelin and 54 ± 4.5 nM for EP-80317.



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  		Figure 5. Effect of 1 µM hexarelin, EP-80317, MK-0677, and human ghrelin on the cell proliferation of CALU-1 human lung carcinoma cell line. Cells (5000/well) were grown for 96 h in the absence (control) or in the presence of the indicated compounds with media changed every 48 h. The cell number was determined at the indicated time by a standardized absorbance assay (violet). Values are mean ± SEM of three separate experiments. a, P < 0.01; b, P < 0.001 vs. control.

 


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  		Figure 6. Dose-response relationship of hexarelin and EP-80317 on the proliferation of CALU-1 lung carcinoma cells. Cells were incubated in the absence (control) or in the presence of increasing concentrations of the indicated peptides, with media changed every 48 h. The cell number was determined at 96 h by a standardized absorbance assay (crystal violet). Values are expressed as a percentage of control (100% = 3.6 ± 0.2 x 104 cells/well) and represent the mean ± SEM of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The peripheral tissue distribution of GHS binding sites labeled by a radioiodinated peptidyl GHS ([125I]Tyr-Ala-hexarelin), which we have studied here, is in agreement with our previous demonstration of the existence of peptidyl GHS binding sites in the human lung tissue (18). We now demonstrate for the first time that these receptors are also present in lung cancer. It will be noted that the specific binding of [125I]Tyr-Ala-hexarelin to membranes of nonendocrine lung carcinomas was significantly greater (about 2-fold higher) than that recorded in nontumoral lung, whereas endocrine lung neoplasms (carcinoid tumors) showed binding values comparable to those of normal lung. These findings suggest that Tyr-Ala-hexarelin binding sites are up-regulated in nonendocrine lung tumors. However, this hypothesis must be considered with caution because additional factors could have reduced the number of these receptors in the normal lung, including a possible postmortem degradation of nontumoral lung tissue (which unlike lung neoplasms, was obtained at autopsy) and/or the presence in this tissue of a cell population less homogeneous than present in lung cancer.

In both normal and neoplastic lung tissue, [125I]Tyr-Ala-hexarelin binding showed properties typical of the ligand-receptor interaction, namely high affinity, saturability, and specificity. The most significant finding was the observation that this binding of the radioligand was inhibited by unlabeled peptidyl GHS such as hexarelin and GHRP-6, but not by ghrelin, or by the nonpeptidyl GHS MK-0677. This implies that the binding sites are specific for peptidyl GHS only and are not likely type 1a GHS receptors. The same binding sites are now reported in CALU-1. Like in nonendocrine lung carcinomas, [125I]Tyr-Ala-hexarelin binding to membranes from CALU-1 cells was completely displaced in a dose-dependent manner by unlabeled peptidyl GHS and EP-80317, a peptidyl GHS analog that is devoid of GH-releasing activity in vivo (Ref. 33 and Locatelli, V., T. Reissmann, I. C. Robinson, personal communications). In contrast, no competition was observed in the presence of MK-0677, ghrelin, as well as of other hormones that have been found to stimulate (GHRH, IGF-II) or inhibit (SRIF-14) the proliferation of endocrine lung cancer cells (34, 35, 36). Thus, the binding properties in CALU-1 cells, as well as in lung neoplastic tissue, overlap with those reported in other nonendocrine normal tissues, including heart, blood vessels, liver, skeletal muscle, kidney, and adipose tissue (18).

By investigating the effects of ghrelin and synthetic GHS on DNA synthesis and growth of CALU-1 cell line, we found that hexarelin and EP-80317, but not ghrelin or MK-0677, inhibited the IGF-II-stimulated thymidine incorporation and cell growth at concentrations close to their binding affinity. These data strongly suggest that the antiproliferative effect of synthetic peptidyl GHS on lung carcinoma cells is not mediated by the GHS-R1a but probably involves specific non-GHS-R1a binding sites. In agreement with this hypothesis is the evidence (present results) that the GHS-R1a is not expressed in either CALU-1 cells, nor in normal lung, nonendocrine lung carcinomas, or in 50% of endocrine neoplasms. Note that this antiproliferative action is also shared by the peptidyl GHS analog EP-80317, which does not have GH-releasing activity in vivo (33). Because EP-80317 is structurally related to the antagonist D-Lys3-GHRP-6, a synthetic hexapeptide that inhibits GH release induced by GHRPs and also by ghrelin (6), we cannot exclude that it may also possess GH-releasing antagonist activity.

The antiproliferative action of peptidyl GHS has already been observed in thyroid tumor cell lines of follicular origin, as well as in breast cancer cell lines (23, 24). Differently from what observed in CALU-1 cell line, this effect in breast carcinoma cells was generally shared by the nonpeptidyl GHS MK-0677 and ghrelin, as well as by the nonacylated form of ghrelin that does not bind the GHS-R1a (8, 9). It will be noted that this antiproliferative effect in mammary carcinoma cells was present despite any significant GHS-R1a mRNA expression (24). At variance with data in breast cancer cells (24), we have now shown that DNA synthesis and proliferation of CALU-1 lung carcinoma cells is inhibited by peptidyl GHS, but not by nonpeptidyl GHS and even by ghrelin. These discrepancies could be due to the presence in the above tumors of different GHS-R subtypes and/or mutated GHS-R that have lost the ability to recognize some, but not other, GHS ligands. This hypothesis is supported by studies demonstrating that a number of closely interrelated GHS-R subtypes exist in various tissues (37, 38) and that some artificial mutants of the GHS-R1a, such as the E124-Q mutant, loose the capacity to bind nonpeptidyl GHS (39). Indeed, it is still unclear whether ghrelin is the sole ligand or one of a number of ligands activating the GHS-R and whether the GHS-R1a used for ghrelin isolation is the sole receptor or one of a group of receptors for such ligands. On account of these uncertainties the intracellular signal transduction mechanisms are still unclear.

In conclusion, this study shows that peptidyl but not nonpeptidyl GHS, as well as ghrelin, inhibit DNA synthesis and proliferation of CALU-1 cell line in vitro. The CALU-1 cells, as well as both nontumoral and tumoral lung tissue, show the presence of a specific receptor that binds synthetic peptidyl GHS only. In clear contrast, GHS-R1a mRNA expression is occasionally found only in endocrine lung neoplasms, but not in other nontumoral or tumoral lung tissues, and it is also absent in the CALU-1 cell line. Therefore, the growth inhibitory effects of peptidyl GHS on CALU-1 cells is likely to be mediated by a specific receptor other than GHS-R1a.


   Acknowledgments
 
We wish to thank Professors Gianni Bussolati, Franco Camanni, and Francesco De Matteis (University of Turin) for their suggestions.


   Footnotes
 
This work was supported in part by grants from the Italian Ministry of University and Research, Rome, Italy (ex-60% 2000 and Cofin 2000 to G.M. and E.G.), Consiglio Nazionale delle Ricerche, Rome, Italy (Grant 98.03040.CT04 to E.G.), Europeptides (Argenteuil, France), and Fondazione Studio delle Malattie Endocrine e Metaboliche (Turin, Italy).

Abbreviations: CALU-1, Human lung carcinoma cell line; GHS, GH secretagogue.

Received July 27, 2001.

Accepted for publication October 26, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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