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Original Studies |
Departments of Biomedical Sciences and Oncology (P.C., M.P., A.S.), Anatomy, Pharmacology and Forensic Medicine (C.G., F.C., G.M.), and Internal Medicine (E.G.), University of Turin, 10126 Turin, Italy; Department of Medical Sciences (A.G.), University of Piemonte Orientale, 28100 Novara, Italy; Cancer Research and Experimental Endocrinology, Asta Medica (T.R.), 60269 Frankfurt am Main, Germany; and Europeptides (R.D.), 95108 Argenteuil, France
Address all correspondence and requests for reprints to: Paola Cassoni, M.D., Department of Biomedical Sciences and Oncology, University of Turin, Via Santena 7, 10126 Turin, Italy. E-mail: paola.cassoni{at}unito.it
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A specific animal and human GHS-receptor (GHS-R) has been cloned (9). It is encoded by a rare messenger RNA (mRNA) with a predicted open reading frame of 366 amino acids with a transmembrane topography typified by the G protein-coupled receptor family (4, 7, 10, 11). Recently, a gastric-derived peptide, named ghrelin, has been proposed as a natural ligand of the GHS receptor (GHS-R) (12). It has been demonstrated that ghrelin has a strong stimulatory effect on GH secretion in the rat (12) and human (13) and displaces [125I]-Tyr-Ala-hexarelin from human pituitary binding sites (14).
The hypothalamus and the pituitary gland show a remarkable density of GHS-R in humans as well as in animals (4, 6, 9, 15). The existence of specific GHS binding sites in the pituitary and central nervous system probably explains endocrine and central activities of GHS (3, 4, 16). However, the GHS-R distribution is not restricted to pituitary or brain. In fact, the expression of type I GHS-R mRNA has been demonstrated in the human pancreas (17) and [125I]-Tyr-Ala-hexarelin labels specific binding sites in the rat and human heart (18, 19, 20), as well as in a wide range of other peripheral human tissues (14, 21). In addition, GHS-Rs were also found in neoplastic tissues, including pituitary adenomas (22), neuroendocrine tumors (23), and thyroid carcinomas of follicular cell origin (in both primary tumors and cell lines) (24).
The normal (nonneoplastic) mammary gland is apparently devoid of specific GHS binding sites, as opposed to other hormonally-regulated glands (14). To our knowledge, no data exist in the literature concerning breast cancer, although it has been reported that nontumoral and neoplastic mammary gland may be regulated by GH-releasing/inhibiting hormones, because specific mRNAs for GHRH and SRIF have been demonstrated in these tissues (25, 26, 27).
Based on the foregoing, the aims of the present study were: 1) to investigate the presence of GHS-R in nontumoral mammary gland and in a series of breast carcinomas and fibroadenomas by means of a radioreceptor assay, using [125I]-Tyr-Ala-hexarelin as tracer; 2) to evaluate the ability of human ghrelin (either octanoylated or desoctanoylated) as well as of other peptidyl and nonpeptidyl GHS (hexarelin, Tyr-Ala-hexarelin, and MK-0677) and analogs (EP-80317 and EP-9399) to compete with the radioligand for binding sites in the above tumors; and 3) to study the effects of these compounds on the proliferation of estrogen-dependent (MCF7, T47D) and estrogen-independent (MDA-MB231) human breast carcinoma cell lines in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human ghrelin (Gly-Ser-Ser-(O n-octanoyl)-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg-NH2), desoctanoyl human ghrelin, MK-0677 (N-[1(R) {[1,2-dihydro-1-methanesulphonylspiro-(3H-indole-3, 4'-piperidin)-1'-yl]-2-(phenyl-methoxy)-ethyl}-2-amino-2-methylpropanamide methane sulphonate], hexarelin (His-D-2Me-Trp-Ala-Trp-D-Phe-Lys-NH2), and three structurally-related analogues 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 EP-9399 [c(Trp-D-Phe-His-2Me-Trp-Ala)] were provided by Europeptides (Argenteuil, France). Human GHRH (GHRH 1–44), SRIF 1–14, human insulin-like growth factor I (IGF-I), and human CRF were purchased from Bachem Feinchemikalien AG (Bubendorf, Switzerland. Tamoxifen and 17-ß estradiol were purchased from Sigma (St. Louis, MO). [125I]-Tyr-Ala-hexarelin (SA 2000 Ci/mmol) was iodinated using a lactoperoxidase method and purified by reverse-phase high-performance liquid chromatography, as previously described (6, 7). [3H]-thymidine (SA 2000 Ci/mmol) was purchased from Amersham Pharmacia Biotech Italia, (Milan, Italy). Penicillin, streptomycin, FCS, trypsin/EDTA solution, and other tissue culture reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD).
Tissue samples
Six normal breast parenchymas (obtained from mammoplasty specimens), 4 breast fibroadenomas and 24 breast carcinomas [17 invasive ductal carcinomas, 2 tubular carcinomas, 5 invasive lobular carcinomas; G1 (n = 8), G2 (n = 8), G3 (n = 8), following World Health Organization grading] were collected from surgical specimens received in the Department of Pathology of the University of Turin in years 1997–1999. No patient was undergoing hormonal (antiestrogenic) treatment. The median age of patients was 62 yr. All patients gave their informed consent for the research use on their tissues, and the study project was approved by our hospital ethical committee. A tissue fragment adjacent to one used for histopathological diagnosis was immediately frozen at -80 C and stored for 4–36 months until processed for membrane preparation and binding studies.
The receptor status (estrogen receptors, ER and progesterone receptors, PgR) and the proliferative index of all breast carcinomas have been evaluated by immunohistochemistry. The following antibodies were used: ER (1D5, diluted 1:100; DAKO Corp., Glostrup, Denmark), PgR (diluted 1:15; BioGenex Laboratories, Inc., San Ramon, CA), ki67 (MIB-1, diluted 1:10; Immunotech, Marseille, France).
Cell cultures
Three immortalized cell lines (MCF7, T47D, and MDA-MB231), derived from human breast carcinomas, were purchased from ATCC (Rockville, MD). Two of them (MCF7 and T47D) were estrogen-dependent, whereas MDA-MB231 was an estrogen-independent cell line. All the cell lines were grown as monolayer in RPMI-1640 medium supplemented with FCS 10% and penicillin/streptomycin in a 5% CO2-humidified atmosphere at 37 C and used in binding and cell proliferation studies.
Binding studies
GHS binding sites were assayed on membranes (30,000 x
g pellet) isolated from human tissues or cell lines, as
previously described (6), using
[125I]-Tyr-Ala-hexarelin as ligand. This
hexarelin analog has been reported to have the same GH-releasing
potency of hexarelin in rats (7) and humans
(28) and to be a reliable probe for labeling human
GHS-R in vitro (6, 7, 14, 24). In preliminary
experiments, it was found that equilibrium conditions for the breast
carcinomas and cell lines were similar to those found for binding to
human hypothalamus and pituitary gland (6). For
single-point binding assay, tissue membranes (corresponding to 100 µg
membrane protein, measured using the method of Lowry et al.)
(29) were incubated in triplicate, at 0 C for 60 min, with
approximately 5 nmol/L [125I]-Tyr-Ala-hexarelin
in a final vol of 0.5 mL assay buffer (50 mmol/L Tris, 2 mmol/L EGTA,
0.1% BSA, 0.03% bacitracin, titrated with HCl to pH 7.3). Parallel
incubations, where 2.5 µmol/L unlabeled Tyr-Ala-hexarelin was also
present, were used to determine nonspecific binding, which was
subtracted from total binding to yield specific binding values. The
binding reaction was terminated by adding ice-cold assay buffer
followed by filtration over Whatman GF/B filters. Filters
were rinsed three times with assay buffer, and the radioactivity bound
to membranes was measured by a Packard auto-
counter. Specific
binding was calculated as the difference between binding in the absence
and in the presence of excess unlabeled Tyr-Ala-hexarelin and 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. 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 Prism 3 program (GraphPad Software, Inc., San Diego, CA). In some assays, receptor binding
saturation studies were also conducted by incubating tissue membranes
with increasing concentrations (from 0.15–20 nmol/L) of radioligand in
the absence and in the presence of a fixed amount (2.5 µmol/L) 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
[maximal binding capacities (Bmax)] were
calculated with the GraphPad Software, Inc. Prism 3
program.
RT-PCR for GHS-R1a
RNA from the MCF7, T47D, and MDA-MB 231 cell lines was extracted by Rnazol (Roche Molecular Biochemicals, Mannheim, Germany) as described by the manufacturer. Then 3 µg RNA was retrotranscribed by Superscript Reverse Transcriptase (Life Technologies, Inc., Roskilde, Denmark) and amplified by AmpliTaq Gold Polymerase (Perkin-Elmer Corp., Foster City, CA). Primers, designed with Primer Express (Perkin-Elmer Corp.), were: 5'-CTCTGCATGCCCCTGGACCTCGTTCGC-3' (forward) and 5'-CTGCCGATGAGACTGTAGAGGACCGTGAGAC-3', which amplifies a 58-bp fragment of GHS-R1a. PCR was carried out by 10 min at 95 C and then by 40 cycles of 15 sec at 95 C and 1 min at 60 C. Amplified DNA was run on 2% agarose electrophoresis, and the image was acquired by Chemidoc (Bio-Rad Laboratories, Inc., Hercules, CA).
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 (31). Briefly, starved breast carcinoma cell lines (2 x 105 cells/mL) were incubated at 37 C, with or without 10% FCS or estradiol (10 nmol/L), in the absence or in the presence of different concentrations (from 1 nmol/L–2 µmol/L) of GHRH, ghrelin, desoctanoyl ghrelin, MK-0677, tamoxifen, and hexarelin and its analogs (EP-80317, EP-9399). After incubation for 20 h, time needed to obtain the maximal effect of all compounds, 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, breast carcinoma cell lines (8 x 103 cells per mL) were seeded out in a 24-well plate containing RPMI medium supplemented with 10% FCS and grown for 96 h in the absence or in the presence of 1 µmol/L GHRH, ghrelin, desoctanoyl ghrelin, tamoxifen, and hexarelin or its analog EP-80317, with media changed every 48 h. In selected experiments, cells were synchronized, 8 h after plating, by a 36-h rest in 0.5% FCS. Cells were detached with trypsin-EDTA solution and counted in double-blind fashion, by two independent investigators, using a hemocytometer. All experiments were done in triplicate.
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, they are expressed 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 the level of significance.
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Results |
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Considerable Tyr-Ala-hexarelin specific binding values were found
in all breast carcinomas (Table 1
). The
highest specific binding activity was observed in well-differentiated
(G1) invasive carcinomas, where it represented about 60–75% of total
radioactivity bound. Tyr-Ala-hexarelin binding was recorded in all
specimens, with binding values that were greater than those previously
found (11.3–14.0%) in a classical GHS target tissue, such as the
human pituitary gland (6). Specific binding was also
present in moderately (G2) and poorly differentiated (G3) ductal
carcinomas, with values that were about 30% and 55% lower than those
detected in G1 carcinomas respectively. Tumor histotype, stage,
receptor status, proliferative index, and hormonal status of patients
(either pre- or postmenopausal) were not correlated to the amount of
binding.
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In contrast, scanty Tyr-Ala-hexarelin binding was observed in the
nontumoral breast parenchyma and in all the fibroadenomas studied
(Table 1
).
Specificity of binding and saturation studies
To determine whether the binding of
[125I]-Tyr-Ala-hexarelin to tissue membranes
shows the properties typical of ligand-receptor interaction, the
binding of radiotracer was investigated in more detail in some
specimens of mammary carcinomas that yielded sufficient amounts of
membranes for these studies and in the MCF7 cells in which the presence
of a specific [125I]-Tyr-Ala-hexarelin binding
was also demonstrated. The specificity of
[125I]-Tyr-Ala-hexarelin binding to membranes
from G1 mammary carcinomas and MCF7 cells was established by
competitive binding experiments, using several natural compounds that
stimulate (human ghrelin, GHRH) or inhibit (SRIF 14) GH secretion, and
some synthetic peptidyl (hexarelin, Tyr-Ala-hexarelin) and nonpeptidyl
(MK-0677) GHS. Various structurally-related analogs of hexarelin
(EP-80317 and EP-9399) and ghrelin (desoctanoyl ghrelin), which do not
have GH-releasing activity in vivo (32, 33 ; and Locatelli V., T. Reissmann, I. C. Robinson,
personal communications), and some hormones (CRF and IGF-I)
functionally unrelated to hexarelin were also studied in these
competitive binding experiments. The binding of
[125I]-Tyr-Ala-hexarelin to membranes of G1
carcinomas was completely displaced by increasing concentrations of
unlabeled hexarelin, Tyr-Ala-hexarelin, EP-80317, MK-0677, ghrelin, and
desoctanoyl ghrelin, whereas none of the structurally and functionally
unrelated peptides (CRF and IGF-I), as well as GHRH, SRIF-14, and
EP-9399 inhibited the binding of radiotracer. Hexarelin,
Tyr-Ala-hexarelin, and EP-80317 exhibited equally high affinity for the
binding sites, whereas MK-0677 and ghrelin, which showed a similar
efficacy to each other, were less effective (3–4 times) than hexarelin
and more potent (4–5 times) than desoctanoyl ghrelin in displacing
[125I]-Tyr-Ala-hexarelin (Fig. 1). The IC50 values
(mean ± SEM of four separate experiments),
all expressed as mol/L x
10-8, were as follows:
hexarelin, 5.3 ± 0.4; Tyr-Ala-hexarelin, 4.3 ± 0.3;
EP-80317, 3.7 ± 0. 4; MK-0677, 15 ± 1; ghrelin, 19 ±
2; and only 80 ± 4 for desoctanoyl ghrelin. The pattern of
displacement specificities in the MCF7 cells resembled that of the
tumor tissue (Fig. 1).
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). Scatchard
analysis of these data (Fig. 2, upper panel) demonstrated
the existence of a single class of high-affinity sites in both breast
carcinomas and MCF7 cells (Kd values were:
1.8 x 10-9 mol/L for
the well-differentiated carcinomas (G1), 1.7 x
10-9 mol/L for the
moderately differentiated carcinomas (G2), 2.6 x
10-9 mol/L for the poorly
differentiated carcinomas (G3), and 1.4 x
10-9 mol/L for the MCF7
cells), with limited binding capacity (Bmax
values were: 2062 fmol/mg protein for the G1 carcinoma, 1057 fmol/mg
protein for the G2 carcinoma, and 882 fmol/mg protein for the G3
carcinoma). The calculated Bmax values of
Tyr-Ala-hexarelin binding sites in three G1 carcinomas were
significantly greater (mean ± SEM of
2198 ± 152 fmol/mg protein) than those measured in less
differentiated neoplasms (1069 ± 99 fmol/mg protein for G2 and
887 ± 131 fmol/mg protein for G3 carcinomas). However, the
Kd values of these three types of tumors were not
substantially different from one another, being (1.8 ± 0.21)
x 10-9 mol/L in the G1
carcinomas, (1.7 ± 0.11) x
10-9 mol/L in the G2
carcinomas, and (2.0 ± 0.26) x
10-9 mol/L in the G3
carcinomas.
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The data presented suggest that a common receptor for ghrelin and
for synthetic GHS may be expressed in mammary carcinomas. Because
either ghrelin and synthetic GHS, peptidic and not peptidic, bind to
GHS-R1a, we have investigated its expression in three different breast
carcinoma cell lines (T47D, MCF7, and MDA-MB231). However, after RT-PCR
(40 cycles), no complementary DNA corresponding to GHS-R1a was obtained
from RNA extracted from mammary cell lines, whereas a significant
amount of complementary DNA was amplified from the same amount of total
RNA extracted from human hypothalamus and peripheral blood lymphocytes
(Fig. 3). These data suggest that the
high-affinity binding sites recognized by hexarelin in these cells
may depend on the expression of a different receptor, which still
awaits to be identified.
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Based on the evidence of specific GHS binding sites in both
primary breast carcinomas and in the three different breast carcinoma
cells (T47D, MCF7, and MDA-MB231) and on the displacement data, we
investigated the effects of human ghrelin, desoctanoyl ghrelin,
MK-0677, GHRH, and hexarelin and its analogues (EP-80317, EP-9399) on
the cell proliferation in vitro. Treatments with tamoxifen,
an antiestrogen that is a first-line drug in the treatment of
estrogen-dependent breast cancer (34), were also included
in this study as negative controls. Both the basal
[3H]-thymidine incorporation (serum-free
conditions) and that stimulated by FCS or estradiol were studied in the
estrogen-dependent MCF7 breast cancer cells. Figure 4 shows that hexarelin, EP-80317,
MK-0677, tamoxifen, ghrelin, and desoctanoyl ghrelin inhibited the
serum-stimulated [3H]-thymidine incorporation
in a concentration-dependent manner and that hexarelin, EP-80317,
MK-0677, and ghrelin were more effective than tamoxifen, whereas
desoctanoyl ghrelin was less effective. The calculated 50% effective
doses (mean ± SEM of four separate
experiments), all expressed as mol/L x
10-8, were as follows:
hexarelin, 4.1 ± 0.8; EP-80317, 3.3 ± 1.0; MK-0677, 14
± 2.0; ghrelin, 16 ± 1. 3; tamoxifen, 26 ± 2.1; and only
81 ± 5.7 for desoctanoyl ghrelin. In contrast, no inhibition was
observed in the presence of some peptides that do not bind to GHS-R,
such as GHRH and EP-9399. In addition, no change in thymidine
incorporation was observed when the different compounds were incubated
with MCF7 cells growing in serum-free conditions.
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), a clear dose-related decrease of
thymidine incorporation was seen only in the presence of tamoxifen
(IC50: 1.0 ± 0.2 x
10-8 mol/L, n = 4),
whereas the other substances studied showed an inhibitory effect only
at highest concentrations (1–2 µmol/L).
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). The simultaneous
treatment with ghrelin and hexarelin did not produce a synergistic
inhibition of cell growth, because the cell number remained unmodified,
compared with the treatment with individual peptides. Similar results
were also found on another estrogen-dependent breast cancer cell
line, T47D (data not shown). When the above compounds were
incubated with an estrogen-receptor negative breast cancer cell line
(MDA-MB231), a significant decrease of cell growth was observed in the
presence of hexarelin, EP-80317, MK-0677, ghrelin, and desoctanoyl
ghrelin, whereas GHRH or tamoxifen were not effective.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a previous study on the GHS-receptor (GHS-R) distribution in peripheral human tissues, we reported that GHS binding sites are not present in the nontumoral mammary gland (14, 21). We have here confirmed these previous observations, because the binding to [125I]-Tyr-Ala-hexarelin was found to be undetectable in the normal breast parenchyma as well as in benign lesions (fibroadenomas). On the contrary, when breast carcinomas were studied, a specific binding for GHS was observed. The entity of the binding was independent from the tumor histological type, stage, ER status, proliferative index, and pre- or postmenopausal age of the patients, but it was directly related to the grade of tumor differentiation. In fact, well-differentiated carcinomas showed a higher GHS binding than moderately and poorly differentiated carcinomas.
An identical profile of GHS binding had been observed in thyroid carcinomas of follicular origin (24). In these tumor tissues, the [125I]-Tyr-Ala-hexarelin binding was reduced as examination moved from well-differentiated carcinomas (i.e. papillary carcinoma) toward the less-differentiated forms (such as poorly differentiated and anaplastic carcinomas) (24). It could therefore be suggested that functionality of (probably overexpressed) GHS binding sites is maintained in better-differentiated tumors (of both thyroid and breast origin) and is decreased in less-differentiated neoplasm.
In the present study in the neoplastic breast tissue, [125I]-Tyr-Ala-hexarelin binding showed properties typical of the ligand-receptor interaction, such as high affinity, saturability, and specificity. The binding of radioligand to membranes of these tissues was inhibited by ghrelin (both octanoylated and desoctanoylated) and various peptidyl and nonpeptidyl GHS. It will be noted that the binding was inhibited even by molecules like desoctanoylated ghrelin and the peptidyl GHS EP-80317, which are devoid of any GH-releasing activity in vivo (32, 33). On the other hand, the binding was unaffected by a number of other peptides (GHRH, SRIF-14, IGF1, and CRF), which are structurally unrelated to peptidyl GHS. These binding properties of breast neoplastic tissue overlap with those reported in endocrine tissues, including pituitary gland, ovary, adrenal, thyroid, and testis (6, 14, 24). However, they are at variance with those generally found in other peripheral nonendocrine tissues that are target for peptidyl GHS (for instance, the heart). In fact, in the latter organs, the specific [125I]-Tyr-Ala-hexarelin binding is weakly inhibited by ghrelin (14)as well as by nonpeptidyl GHS (14, 19, 35). Taken together, these data support the hypothesis that different GHS binding site subtypes may exist in peripheral organs, possibly depending on their endocrine or nonendocrine nature (9, 10, 20) but also on their normal or neoplastic nature (present study).
Specific binding for GHS, showing the same properties recorded in neoplastic breast tissue, was demonstrated also in three human breast carcinoma cell lines either estrogen-dependent (MCF7 and T47D) or -independent (MDA-MB231). In all these cell lines, GHS-R1a mRNA was not detected by RT-PCR. This implies that the specific GHS binding sites in these tissues are different from the classical GHS-R1, as well as from the specific binding sites characterized in the cardiovascular system (14, 18, 19, 35). Theoretically, the binding sites we have now described in the neoplastic breast tissue could be the same ones we have previously demonstrated in the normal and tumoral human thyroid tissue (24).
In agreement with this hypothesis, in the present study, GHS were found able to inhibit proliferation of breast carcinoma cells, as previously reported for thyroid carcinoma cell lines (24). Both octanoylated and desoctanoylated human ghrelin, as well as peptidyl and nonpeptidyl GHS, were able to inhibit serum-stimulated cell growth and thymidine incorporation at concentrations close to their binding affinity. Again notice that thymidine incorporation and cell proliferation were inhibited even by molecules like desoctanoylated ghrelin and the hexarelin derivative EP-80317, which are devoid of any GH-releasing activity in vivo (32, 33).
Interestingly, GHS were even found to be able to counteract the estradiol-stimulated thymidine incorporation, though high concentrations were required to obtain this effect. Moreover, in estrogen-independent MDA-MB231 cells, GHS and analogs are able to inhibit cell proliferation, whereas tamoxifen is ineffective.
In conclusion, this study shows that natural and synthetic GHS possess biological activities that are independent of their well-recognized GH-releasing activity. Thus, inhibition of breast cancer cell growth was induced even by desoctanoylated ghrelin and EP-80317, which possess no stimulatory effect on GH secretion (32, 33). The availability of molecules able to counteract tumor cell growth without any stimulatory effect on GH and PRL release could, theoretically, have potential clinical applications. In progress are experiments aimed at evaluating, in vivo, the antiproliferative effect of the different GHS and analogs in both estrogen-dependent and -independent tumors, mainly focusing on hexarelin derivatives devoid of GH-releasing properties, and at clarifying the intracellular mechanisms involved.
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Acknowledgments |
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Footnotes |
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Received August 4, 2000.
Revised November 20, 2000.
Accepted December 18, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 N. Filigheddu, V. F. Gnocchi, M. Coscia, M. Cappelli, P. E. Porporato, R. Taulli, S. Traini, G. Baldanzi, F. Chianale, S. Cutrupi, et al. Ghrelin and Des-Acyl Ghrelin Promote Differentiation and Fusion of C2C12 Skeletal Muscle Cells Mol. Biol. Cell, March 1, 2007; 18(3): 986 - 994. [Abstract] [Full Text] [PDF]
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K. Wagner, K. Hemminki, E. Grzybowska, R. Klaes, B. Burwinkel, P. Bugert, R. K. Schmutzler, B. Wappenschmidt, D. Butkiewicz, J. Pamula, et al. Polymorphisms in genes involved in GH1 release and their association with breast cancer risk Carcinogenesis, September 1, 2006; 27(9): 1867 - 1875. [Abstract] [Full Text] [PDF]
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 V. D. Dixit, A. T. Weeraratna, H. Yang, D. Bertak, A. Cooper-Jenkins, G. J. Riggins, C. G. Eberhart, and D. D. Taub Ghrelin and the Growth Hormone Secretagogue Receptor Constitute a Novel Autocrine Pathway in Astrocytoma Motility J. Biol. Chem., June 16, 2006; 281(24): 16681 - 16690. [Abstract] [Full Text] [PDF]
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K. H. Paik, Y. H. Choe, W. H. Park, Y. J. Oh, A. H. Kim, S. H. Chu, S. W. Kim, E. K. Kwon, S. J. Han, W. Y. Shon, et al. Suppression of Acylated Ghrelin during Oral Glucose Tolerance Test Is Correlated with Whole-Body Insulin Sensitivity in Children with Prader-Willi Syndrome J. Clin. Endocrinol. Metab., May 1, 2006; 91(5): 1876 - 1881. [Abstract] [Full Text] [PDF]
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W. Wei, X. Qi, J. Reed, J. Ceci, H.-Q. Wang, G. Wang, E. W. Englander, and G. H. Greeley Jr. Effect of chronic hyperghrelinemia on ingestive action of ghrelin Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2006; 290(3): R803 - R808. [Abstract] [Full Text] [PDF]
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T Akamizu, T Murayama, S Teramukai, K Miura, I Bando, T Irako, H Iwakura, H Ariyasu, H Hosoda, H Tada, et al. Plasma ghrelin levels in healthy elderly volunteers: the levels of acylated ghrelin in elderly females correlate positively with serum IGF-I levels and bowel movement frequency and negatively with systolic blood pressure J. Endocrinol., February 1, 2006; 188(2): 333 - 344. [Abstract] [Full Text] [PDF]
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 P L Jeffery, R E Murray, A H Yeh, J F McNamara, R P Duncan, G D Francis, A C Herington, and L K Chopin Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines Endocr. Relat. Cancer, December 1, 2005; 12(4): 839 - 850. [Abstract] [Full Text] [PDF]
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 A. P. Davenport, T. I. Bonner, S. M. Foord, A. J. Harmar, R. R. Neubig, J.-P. Pin, M. Spedding, M. Kojima, and K. Kangawa International Union of Pharmacology. LVI. Ghrelin Receptor Nomenclature, Distribution, and Function Pharmacol. Rev., December 1, 2005; 57(4): 541 - 546. [Abstract] [Full Text] [PDF]
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 A. H. Yeh, P. L. Jeffery, R. P. Duncan, A. C. Herington, and L. K. Chopin Ghrelin and a Novel Preproghrelin Isoform Are Highly Expressed in Prostate Cancer and Ghrelin Activates Mitogen-Activated Protein Kinase in Prostate Cancer Clin. Cancer Res., December 1, 2005; 11(23): 8295 - 8303. [Abstract] [Full Text] [PDF]
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 A. P Silva, K. Bethmann, F. Raulf, and H. A Schmid Regulation of ghrelin secretion by somatostatin analogs in rats Eur. J. Endocrinol., June 1, 2005; 152(6): 887 - 894. [Abstract] [Full Text] [PDF]
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 M. Groschl, H. G. Topf, J. Bohlender, J. Zenk, S. Klussmann, J. Dotsch, W. Rascher, and M. Rauh Identification of Ghrelin in Human Saliva: Production by the Salivary Glands and Potential Role in Proliferation of Oral Keratinocytes Clin. Chem., June 1, 2005; 51(6): 997 - 1006. [Abstract] [Full Text] [PDF]
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F. Gaytan, C. Morales, M. L. Barreiro, P. Jeffery, L. K. Chopin, A. C. Herington, F. F. Casanueva, E. Aguilar, C. Dieguez, and M. Tena-Sempere Expression of Growth Hormone Secretagogue Receptor Type 1a, the Functional Ghrelin Receptor, in Human Ovarian Surface Epithelium, Mullerian Duct Derivatives, and Ovarian Tumors J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1798 - 1804. [Abstract] [Full Text] [PDF]
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W. Wei, G. Wang, X. Qi, E. W. Englander, and G. H. Greeley Jr. Characterization and Regulation of the Rat and Human Ghrelin Promoters Endocrinology, March 1, 2005; 146(3): 1611 - 1625. [Abstract] [Full Text] [PDF]
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C. Gauna, P. J. D. Delhanty, L. J. Hofland, J. A. M. J. L. Janssen, F. Broglio, R. J. M. Ross, E. Ghigo, and A. J. van der Lely Ghrelin Stimulates, Whereas Des-Octanoyl Ghrelin Inhibits, Glucose Output by Primary Hepatocytes J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1055 - 1060. [Abstract] [Full Text] [PDF]
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H. Ariyasu, K. Takaya, H. Iwakura, H. Hosoda, T. Akamizu, Y. Arai, K. Kangawa, and K. Nakao Transgenic Mice Overexpressing Des-Acyl Ghrelin Show Small Phenotype Endocrinology, January 1, 2005; 146(1): 355 - 364. [Abstract] [Full Text] [PDF]
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 G. Rindi, A. Torsello, V. Locatelli, and E. Solcia Ghrelin Expression and Actions: A Novel Peptide for an Old Cell Type of the Diffuse Endocrine System Experimental Biology and Medicine, November 1, 2004; 229(10): 1007 - 1016. [Abstract] [Full Text] [PDF]
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M. L. Barreiro, F. Gaytan, J. M. Castellano, J. S. Suominen, J. Roa, M. Gaytan, E. Aguilar, C. Dieguez, J. Toppari, and M. Tena-Sempere Ghrelin Inhibits the Proliferative Activity of Immature Leydig Cells in Vivo and Regulates Stem Cell Factor Messenger Ribonucleic Acid Expression in Rat Testis Endocrinology, November 1, 2004; 145(11): 4825 - 4834. [Abstract] [Full Text] [PDF]
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 A. J. van der Lely, M. Tschop, M. L. Heiman, and E. Ghigo Biological, Physiological, Pathophysiological, and Pharmacological Aspects of Ghrelin Endocr. Rev., June 1, 2004; 25(3): 426 - 457. [Abstract] [Full Text] [PDF]
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N. M. Neary, C. J. Small, A. M. Wren, J. L. Lee, M. R. Druce, C. Palmieri, G. S. Frost, M. A. Ghatei, R. C. Coombes, and S. R. Bloom Ghrelin Increases Energy Intake in Cancer Patients with Impaired Appetite: Acute, Randomized, Placebo-Controlled Trial J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 2832 - 2836. [Abstract] [Full Text] [PDF]
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 M. J Iglesias, R. Pineiro, M. Blanco, R. Gallego, C. Dieguez, O. Gualillo, J. R Gonzalez-Juanatey, and F. Lago Growth hormone releasing peptide (ghrelin) is synthesized and secreted by cardiomyocytes Cardiovasc Res, June 1, 2004; 62(3): 481 - 488. [Abstract] [Full Text] [PDF]
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 A. INUI, A. ASAKAWA, C. Y. BOWERS, G. MANTOVANI, A. LAVIANO, M. M. MEGUID, and M. FUJIMIYA Ghrelin, appetite, and gastric motility: the emerging role of the stomach as an endocrine organ FASEB J, March 1, 2004; 18(3): 439 - 456. [Abstract] [Full Text] [PDF]
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J. P. Camina, M. C. Carreira, S. El Messari, C. Llorens-Cortes, R. G. Smith, and F. F. Casanueva Desensitization and Endocytosis Mechanisms of Ghrelin-Activated Growth Hormone Secretagogue Receptor 1a Endocrinology, February 1, 2004; 145(2): 930 - 940. [Abstract] [Full Text] [PDF]
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F. Gaytan, M. L. Barreiro, J. E. Caminos, L. K. Chopin, A. C. Herington, C. Morales, L. Pinilla, R. Paniagua, M. Nistal, F. F. Casanueva, et al. Expression of Ghrelin and Its Functional Receptor, the Type 1a Growth Hormone Secretagogue Receptor, in Normal Human Testis and Testicular Tumors J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 400 - 409. [Abstract] [Full Text] [PDF]
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N. M. Thompson, D. A. S. Gill, R. Davies, N. Loveridge, P. A. Houston, I. C. A. F. Robinson, and T. Wells Ghrelin and Des-Octanoyl Ghrelin Promote Adipogenesis Directly in Vivo by a Mechanism Independent of the Type 1a Growth Hormone Secretagogue Receptor Endocrinology, January 1, 2004; 145(1): 234 - 242. [Abstract] [Full Text] [PDF]
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 Y.-T. Shen, J. J. Lynch, R. J. Hargreaves, and R. J. Gould A Growth Hormone Secretagogue Prevents Ischemic-Induced Mortality Independently of the Growth Hormone Pathway in Dogs with Chronic Dilated Cardiomyopathy J. Pharmacol. Exp. Ther., August 1, 2003; 306(2): 815 - 820. [Abstract] [Full Text] [PDF]
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K. Tanaka, H. Minoura, T. Isobe, H. Yonaha, H. Kawato, D. F. Wang, T. Yoshida, M. Kojima, K. Kangawa, and N. Toyoda Ghrelin Is Involved in the Decidualization of Human Endometrial Stromal Cells J. Clin. Endocrinol. Metab., May 1, 2003; 88(5): 2335 - 2340. [Abstract] [Full Text] [PDF]
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I. Depoortere, T. Thijs, L. Thielemans, P. Robberecht, and T. L. Peeters Interaction of the Growth Hormone-Releasing Peptides Ghrelin and Growth Hormone-Releasing Peptide-6 with the Motilin Receptor in the Rabbit Gastric Antrum J. Pharmacol. Exp. Ther., May 1, 2003; 305(2): 660 - 667. [Abstract] [Full Text] [PDF]
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M. K. Reimer, G. Pacini, and B. Ahren Dose-Dependent Inhibition by Ghrelin of Insulin Secretion in the Mouse Endocrinology, March 1, 2003; 144(3): 916 - 921. [Abstract] [Full Text] [PDF]
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 M. Volante, E. Allia, E. Fulcheri, P. Cassoni, E. Ghigo, G. Muccioli, and M. Papotti Ghrelin in Fetal Thyroid and Follicular Tumors and Cell Lines: Expression and Effects on Tumor Growth Am. J. Pathol., February 1, 2003; 162(2): 645 - 654. [Abstract] [Full Text] [PDF]
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 G. Baldanzi, N. Filigheddu, S. Cutrupi, F. Catapano, S. Bonissoni, A. Fubini, D. Malan, G. Baj, R. Granata, F. Broglio, et al. Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT J. Cell Biol., December 23, 2002; 159(6): 1029 - 1037. [Abstract] [Full Text] [PDF]
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 M. Volante, E. Fulcheri, E. Allia, M. Cerrato, A. Pucci, and M. Papotti Ghrelin Expression in Fetal, Infant, and Adult Human Lung J. Histochem. Cytochem., August 1, 2002; 50(8): 1013 - 1021. [Abstract] [Full Text] [PDF]
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F. Broglio, E. Arvat, A. Benso, C. Gottero, F. Prodam, S. Grottoli, M. Papotti, G. Muccioli, A. J. van der Lely, R. Deghenghi, et al. Endocrine Activities of Cortistatin-14 and Its Interaction with GHRH and Ghrelin in Humans J. Clin. Endocrinol. Metab., August 1, 2002; 87(8): 3783 - 3790. [Abstract] [Full Text] [PDF]
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M. Volante, E. AllIa, P. Gugliotta, A. Funaro, F. Broglio, R. Deghenghi, G. Muccioli, E. Ghigo, and M. Papotti Expression of Ghrelin and of the GH Secretagogue Receptor by Pancreatic Islet Cells and Related Endocrine Tumors J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1300 - 1308. [Abstract] [Full Text] [PDF]
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C. Ghe, P. Cassoni, F. Catapano, T. Marrocco, R. Deghenghi, E. Ghigo, G. Muccioli, and M. Papotti The Antiproliferative Effect of Synthetic Peptidyl GH Secretagogues in Human CALU-1 Lung Carcinoma Cells Endocrinology, February 1, 2002; 143(2): 484 - 491. [Abstract] [Full Text] [PDF]
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M. Papotti, P. Cassoni, M. Volante, R. Deghenghi, G. Muccioli, and E. Ghigo Ghrelin-Producing Endocrine Tumors of the Stomach and Intestine J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 5052 - 5059. [Abstract] [Full Text] [PDF]
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