Ghrelin's role on gastrointestinal tract cancer

Surgical Oncology (2010) 19, e2ee10 available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/suronc

REVIEW

Ghrelin’s role on gastrointestinal tract cancer Dimitrios Nikolopoulos a,*, Stamatis Theocharis b, Gregory Kouraklis a a b

2nd Department of Propedeutic Surgery, University of Athens, Medical School, Laiko General Hospital, Athens, Greece Department of Forensic Medicine and Toxicology, University of Athens, Medical School, Athens, Greece

Accepted 14 February 2009

KEYWORDS Ghrelin; Growth hormone secretagogues (GHSs); Ghrelin receptor or receptor of GHSs (GHS-R); Gastrointestinal tract cancer

Abstract Ghrelin is a recently identified 28-amino-acid peptide, with pituitary growth hormone releasing activities in humans and other mammals. In mammals, ghrelin plays a variety of roles, including influence on food intake, gastric motility, and acid secretion of the gastrointestinal tract. It is mainly secreted from the stomach mucosa, but it is also expressed widely in other tissues e in normal and malignant conditions e and, therefore, ghrelin may exert such variable endocrine and paracrine effects, as autocrine and/or paracrine function in cancer. Ghrelin’s actions are mediated via its receptor, known as growth hormone secretagogue receptor (GHS-R), type 1a and 1b. Several endocrine and non-endocrine cancers, such as gastro-entero-pancreatic carcinoids, colorectal neoplasms, pituitary adenomas, pulmonary and thyroid tumours, as well as lung, breast, and pancreatic carcinomas express ghrelin at both mRNA and protein levels. In the current review, we summarise the available so far data with regard to: (a) the structure of the ghrelin molecule and its receptor; (b) its tissue contribution in physiologic and neoplasmatic conditions; and (c) ghrelin’s possible role in carcinogenesis; specifically, in the area of gastrointestinal tract cancer. The aim of the present study is to determine whether or not ghrelin promotes the proliferation rate of the gastrointestinal tract (GIT) tumours. ª 2009 Elsevier Ltd. All rights reserved.

Abbreviations: GIT, gastrointestinal tract; GHS-R, GH secretagogue receptor; GH, growth hormone; GHRPs, growth hormone releasing peptides; GHSs, growth hormone secretagogues; GHRH, growth hormone releasing hormone; HPLC, high pressure liquid chromatography; VMN, ventromedial nucleus; DMN, dorsomedial nucleus; PVN, paraventricular nucleus; RT-PCR, reverse transcription polymerase chain reaction; IHC, immunohistochemistry. * Corresponding author. Theotokopoulou 29 str, Metamorfosi Attikis, 14452, Greece. Tel.: þ30 69 4483 7783; fax: þ30 210 777 5354. E-mail address: [email protected] (D. Nikolopoulos). 0960-7404/$ - see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.suronc.2009.02.011

Ghrelin’s Role on Gastrointestinal Tract Cancer

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e3 Structure, tissue expression and distribution of ghrelin molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e3 The role of ghrelin axis in malignancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e4 Ghrelin and gastrointestinal tract cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e7 Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e8 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .e8

Introduction In 1976, Bowers and Momany in their effort to identify more powerful and less addictive opiate narcotics, reported that growth hormone (GH) was specifically released in vitro, by synthetic peptide analogues of the opiate met-encephalin [1e3]. That report initiated a search for many other synthetic peptide analogues with potent GH releasing efficacy and limited or no opiate effect; as a result, growth hormone releasing peptides (GHRPs) were discovered. In 1996, Smith et al. identified a specific G-protein coupled receptor, the growth hormone secretagogues receptor (GHS-R), which expressed mainly in the hypothalamus, pituitary and hippocampus and where GHS molecules bound and exerted GH releasing activity [4e7]. This was soon followed, in 1999, by the discovery of an endogenous ligand for this receptor: ghrelin [8]. The discovery of ghrelin is an example of reverse pharmacology, since starting from GHSs, researchers identified the natural ghrelin receptor (GHS-R) and later it ended with the discovery of its natural ligand, ghrelin [9,10]. Ghrelin is a 28-amino-acid orexigenic peptide, which is primarily produced and secreted by the stomach. Ghrelincontaining cells are found to be a distinct endocrine cell type in the submucosal layer of the stomach, known as X/A like cells, which represent a major endocrine population in the oxyntic mucosa. Surprisingly, ghrelin is also present in small amounts in the hypothalamus and pituitary, and, therefore, represents a new member of the brain-gut peptide family. Ghrelin causes robust stimulation of GH secretion [3,4,9]. Although it was known that GH release from the pituitary was controlled tightly by the hypothalamic growth hormone releasing hormone (GHRH); exogenous administration of GHSs induced GH release through a different pathway. Indeed, GHRH acts on the GHRH receptor and consequently increases intracellular cAMP; whereas GHSs act on a different receptor and increase the intracellular calcium (Caþ2) concentration via the inositol 1,4,5-triphosphate signal transduction pathway [6]. Since its discovery, ghrelin has been implicated in wide range of physiological and pathophysiological activities including the control of energy metabolism and cell proliferation.

Structure, tissue expression and distribution of ghrelin molecule In December 1999, Kojima et al., reported for the first time the identification of an endogenous ligand for the former

orphan receptor GHS-R1a. In that ligand was given the name ghrelin from the Proto-Indo-European word ‘‘ghre’’, which means grow, and ‘‘relin’’ because it had GH releasing activities [8,11]. Ghrelin is a 28-amino-acid peptide with a fatty acid chain modification on the N-terminal third amino-acid, Serine (Ser3). High pressure liquid chromatography (HPLC) and mass spectrometry were used to identify the amino-acid sequence of ghrelin and a discrepancy between the observed and calculated molecular weight pointed to the presence of a post-translational modification. The hydroxyl group of the N-terminal end of Ser3 is replaced by a hydrophobic segment, C7H15CO; in other words, the hydroxyl group of Ser3 is octanoylated. This esterification takes place into ghrelin cell’s cytosome; and hereupon the acylated form is secreted in the systematic circulation. Esterification is accomplished almost in 80% of the ghrelin produced in humans [6,11]. In mammals, no other naturally occurring peptide has been previously shown to have this acyl-group as a posttranslational modification [12]. The n-octanoyl group at Ser3 of the ghrelin molecule seems to be essential for some of its endocrine, autocrine or paracrine actions, including GH release, appetite and cell proliferation [7,9]. Furthermore, this post-translational acyl-modification is speculated to confer hydrophobicity upon the N-terminus of ghrelin, allowing it to enter the brain barrier and thereby target the hypothalamus/pituitary system [13]. There may also be a hydrophobic interaction between the n-octanoyl group and the GHS-R receptor that is important for molecular recognition [14]. On the other hand, non-acylated ghrelin (desoctanoyl or desacyl) circulates in far greater amounts than the acylated form and does not displace ghrelin from its hypothalamic and pituitary binding sites, and nor is it able to stimulate GH release in vivo, in rats and humans [15]. However, increasing numbers of studies report such biological effects of desoctanoyl ghrelin as cardiovascular actions and anti-proliferating actions in cancer [16e19]. The ghrelin gene in humans is located on chromosome 3 (3p25e26) [20]. Ghrelin was firstly isolated in the stomach where a number of different types of endocrine cells, exist. To date, four types of endocrine cells, histamine-rich enterochromographin (ECL), somatostatin producing (D), serotonin producing enterochromaffin (EC), and X/A-like, cells have been identified in the oxyntic mucosa by means of ultrastructural and immunohistochemical criteria. The relative percentages of these four cell types in rat oxyntic gland are 60e70% for ECL, 20% for X/A-like, 2e5% for D, and 0e2% EC cells; those in human oxyntic gland are 30% for

e4 ECL, 20% for X/A-like, 22% for D, and 7% for EC cells. The ghrelin-producing cells are equivalent to those previously known as A or X/A-like cells, whose hormonal product was previously unidentified [21,22]. In the stomach, ghrelin cells are round or ovoid; and with high cytoplasm content in secreting cystidiums (granules). These granules are round, solid and thin-walled, while their diameter differs between species [23]. Ghrelin cells in the stomach are not in contact with the lumen but positioned close to the capillaries; that’s why ghrelin is not secreted in the gastrointestinal tract as the other peptide enzymes are, but it is secreted into the vascular system and then, through systematic circulation, transferred to the whole body [24]. Ghrelin is found in the oxyntic mucosa with moderate amounts also in antrum and duodenum [22]. A smaller number of positive on ghrelin cells are found in the small and large intestine, in both humans and rats [2,21]. It has been suggested that the majority of circulating ghrelin (two-thirds) is produced by the stomach, while a smaller proportion (30%) originates from the small intestine [25,26]. In the lower gastrointestinal tract, two types of ghrelin-secreting cells have been observed: the first, the so-called closed cells, have no contact with the lumen, and the elongated open cells which have contact with the lumen (as in the stomach) [27]. The circulating blood ghrelin levels found in a rats blood circulation following surgical removal of the acid producing part of the stomach, decreased by 80%, suggesting that the oxyntic mucosa is the major source of ghrelin [22]. A similar drop of plasma ghrelin levels was found after gastrectomy (~65%) [4] or following gastric by-pass surgery, in humans [28]. Apart from the intestinal tract, ghrelin expression has been identified in a variety of tissues, at mRNA or protein level, or both. Ghrelin peptide has been shown to be expressed in the hypothalamus (arcuate nucleus), in the internuclear space between the lateral hypothalamus, in the ventromedial nucleus (VMN), in the dorsomedial nucleus (DMN), in the paraventricular nucleus (PVN), and in the ependymal layer of the third ventricle [29]; also in the amygdale, outside the hypothalamus on the bed nucleus of the stria terminalis, in the thalamus, hypophysis, Edingere Westphal nucleus, hippocampus, in the medulla oblongata, in the cerebellum and in the habenula [29e32]. Ghrelin mRNA expression has been described to varying degrees in all normal human tissues, as well as in the immunological system [33,34], in the lungs [35], and in the placenta [36]; it has been suggested that it presents cyclic expression in the ovaries [37,38], and has been identified in the testes and the kidneys [39,40]. In the pancreas, some researchers have presented ghrelin in b-insular cells of Langerhans of humans, where ghrelin was co-expressed with insulin [41]; some other investigators have presented ghrelin in human a-insular cells of Langerhans, where ghrelin was co-expressed with glucagon [42], while in other studies, in a novel characterised group of insular cells, the so-called e-cells, where only ghrelin was expressed [43,44].

The role of ghrelin axis in malignancy Is ghrelin carcinogenic or protective against malignancy? Can ghrelin promote cell proliferation and differentiation in

D. Nikolopoulos et al. malignant tissues or can inhibit proliferation and protect against cancer? There are multiple studies with controversial results that should merit discussion in this review article. That’s why an endocrine, autocrine or paracrine role of ghrelin in neoplasia has been suggested in a variety of tumours (Table 1) [17,45e54,56]. First of all, there is now increasing evidence supporting a role for the ghrelin/GHS-R axis in cancer, possibly via autocrine/paracrine mechanisms similar to those seen with GHRH. GHS-R1a and 1b mRNA isoforms are co-expressed in somatotroph, mammosomatotroph, lactotroph and corticotroph adenomas. Ghrelin is also expressed at the mRNA and protein levels in normal and adenomatous human pituitary tissue, indicating that the ghrelin may have a modulatory role in GH release in both the normal and neoplastic pituitary gland. The co-expression of ghrelin and the GHS-R in human pituitary adenomas has led to speculation that ghrelin produced by them, may contribute to their development [19,30]. The highest level of ghrelin mRNA, was expressed in non-functioning adenomas, followed by GH-producing and gonadotrophin-producing (LH/ FSH) ones, while prolactinomas appeared to express the lowest level [45]. On the other hand, corticotropeproducing adenomas were negative for ghrelin in their cytosomes. The initial results suggest that ghrelin produced in pituitary adenoma may play some role in the mechanism underlying its development through autocrine and/or paracrine pathways [45]. The only in vitro study that has presented ghrelin’s proliferative effect on pituitary cell lines is that of Nanzer et al., published in 2004. To be more specific, the researchers provided novel evidence that ghrelin stimulates proliferation of the GH3 pituitary somatotroph tumour cell line, at 1010e106 M concentrations. The effect of ghrelin on cell proliferation was studied by using [3H]-thymidine incorporation; cell counting, whereas the researchers also investigated the activation of the MAPK pathway by using immunoblotting and inhibitors of the extracellular signalregulated kinase 1 and 2 (ERK 1/2), protein kinase C (PKC) and tyrosine phosphatase pathways. Ghrelin caused a significant increase in phosphorylated ERK 1/2, while the positive effect of ghrelin on [3H]-thymidine incorporation was abolished by the MAPK kinase inhibitor U0126, the PKC inhibitor GF109203X and the tyrosine kinase inhibitor tyrphostin 23, suggesting that the ghrelin-induced cell proliferation of GH3 cells is mediated both via a PKC-MAPKdependent pathway and via a tyrosine kinase-dependent pathway [46]. Later on, researchers, as Jeffery et al., Cassoni et al., Papotti et al. by investigating ghrelin’s possible actions in different cancer cell lines and in neuroendocrine tumours, suggesting for the first time that ghrelin promotes malignancy. By using RT-PCR, specific binding sites for ghrelin and GHSs were identified in human malignant thyroid, lung and prostate cell lines, and in benign prostate hypertrophy [47e50]. More specifically, Cassoni et al. suggested that ghrelin and GHSs produced in human medullary thyroid carcinoma, stimulated cell proliferation after 96 h of exposure in ghrelin; in contrast to other forms of thyroid cancer [anaplastic (ARO), papillary (NPA) or follicular (WRO) carcinoma], where ghrelin and GHSs inhibited cell proliferation effect [47]. Volante et al. have also

Ghrelin’s Role on Gastrointestinal Tract Cancer Table 1

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Is ghrelin carcinogenic or protective against malignancy?.

Type of malignant or benign cell lines

Effect on cell proliferation

Reference

HepG2 hepatoma cells Human breast cancer (oestrogen-dependent & independent cell lines) Human breast cancer (oestrogen-dependent & independent cell lines) Human lung carcinoma cell line (CALU-1) Human prostate cancer (ALVA-41, LNCaP, DU145 and PC3 cell lines) Human prostate cancer (DU-145, PC-3 and LNCaP cell lines)

[ [ (110e120 pM) Y (1 mM) Y [ DU-145 Y PC-3 (10e100 pmol/l) [ PC-3 (1 nmol/lt-1 mmol/l) Y LNCaP (no response) [ [ Y

Murata et al. [52] Jeffery et al. [55] Cassoni et al. [17] Ghe et al. [49] Jeffery et al. [51] Cassoni et al. [50]

Y [ [ [

Volante et al. [48] Waseem et al. [53] Waseem et al. [74] Delhanty et al. [57]

Pituitary adenomas cancer (rat GH3 pituitary somatotroph tumour cells) Human medullary thyroid cancer Human papillary (NPA) or anaplastic (ARO) or follicular (WRO) thyroid cancer Follicular thyroid cancer (N-PAP and ARO carcinoma cell lines) Pancreatic adenocarcinomas (PANC1) Colorectal cancer Adrenocortical carcinoma (cell line SW-13)

Nanzer et al. [46] Cassoni et al. [47] Cassoni et al. [47]

According to the studies, ghrelin could inhibit cell proliferation in vitro -as in papillary thyroid cancer and lung cancer- whereas in other neoplastic cells, ghrelin could promote proliferation -such as in breast, prostate and colorectal cancer. Lastly, still have been published studies where it is controversial whether ghrelin is truly carcinogenic or protective against malignancy.

presented, by in vitro studies, a ghrelin’s specific, dosedependent anti-proliferative effect in follicular-derived thyroid carcinoma cell lines (N-PAP and ARO) [48]. Moreover, Jeffery et al. by investigating the expression of the ghrelin axis in breast cancer tissues and by examining the effect of ghrelin on breast cancer cell proliferation in vitro observed that it plays a role in carcinogenesis by stimulating cell proliferation. That study was the first to report the expression of a new exon 3-deleted preproghrelin variant in the breast that was more highly expressed in breast malignant compared with normal tissues. It was also, for the first time, reported that GHSR1b is differentially expressed in normal compared with malignant breast tissue specimens. Moreover, they demonstrated the expression of ghrelin, exon 3-deleted preproghrelin, and GHS-R1a and 1b in the both oestrogendependent and independent malignant cell lines and in the non-tumourigenic benign breast epithelial cell line. The potential tumour-promoting role of ghrelin was supported by the finding that ghrelin increases proliferation in breast cancer cell lines. The exogenous addition by n-octanoylated ghrelin, significantly stimulated the proliferation of the highly metastatic MDA-MB-435 cell line (up to 36% over controls) and in the oestrogen-independent MDA-MB-231 (up to 17% over controls) breast cancer cell lines [51]. In contrast to Jeffery’s et al. study, Cassoni et al. documented an antiproliferative effect of ghrelin in human breast cancer cells [17]. In their study, specific binding sites for ghrelin and GHSs in human breast cancer cell lines were noted. The entity of the binding was independent of the tumour histological type, stage, ER status, proliferative index, and the pre- or postmenopausal age of the patients, but it was directly related to the grade of tumour differentiation. More specifically, high binding potency was observed in well differentiated invasive breast carcinomas, while there was down-regulation of the binding potency in

middle to poor differentiated ones. GHS-R receptors existed in both oestrogen-dependent (MCF7 & T47D) and oestrogen-independent (MDA-MB-231) breast cancer cell lines. Ghrelin and GHSs (peptidyl and non-peptidyl) inhibited cellular proliferation in vitro, independent of the GH releasing activity [17]. The differences observed between these two studies could be attributed to the different ghrelin concentration used; as Jeffery et al. used in vitro the normal serum concentration of ghrelin (110e120 pM) [51], whereas Cassoni et al. used much more high concentration (1 mM), which is believed to be possibly cytotoxic [17]. Furthermore, in another study of Jeffery et al., it was suggested a potential tumour-promoting role of ghrelin in prostate cancer; as it was observed in vitro that ghrelin stimulated (ALVA-41, LNCaP, DU145 and PC3) prostate cancer cells proliferation (almost 33%) [52,53]. Two years later, Cassoni et al. presented that, in androgen-independent prostate cancer cell lines (PC-3) and in benign prostate hypertrophy, ghrelin augmented the cell proliferation in low concentrations (10 pmol/l), whereas in high concentrations there was down-regulation. Furthermore, in androgen-dependent prostate cancer cell lines (DU-145), it was observed inhibition of the cell proliferation, independent of ghrelin concentration status [50]. The exact mechanisms behind this proliferative response have yet to be identified. Ghrelin may induce endogenous GH secretion from the prostate which, in turn, triggers autocrine IGF-I production and secretion. Alternatively, activation of the GHS-R and consequent signaling pathways may itself result in cell proliferation [51]. Two other studies have attested ghrelin’s possible carcinogenetic action are that of Murata etal. and Waseem et al. More specifically Murata reported that ghrelin activated cell proliferation in hepatoma cells [54], while Waseem investigated ghrelin’s action in pancreatic

e6 adenocarcinomas and observed that it promoted cellular proliferation and invasiveness. On the one hand researchers demonstrated expression of the GHS-R1a and 1b isoforms in pancreatic adenocarcinomas cells; on the other hand, although ghrelin transcript was only weakly present in the poorly differentiated human pancreatic cancer cell line, PANC1, and ghrelin peptide was not detected, cellular proliferation, invasiveness, and motility were stimulated by exposure to 10 nM ghrelin, indicating that pancreatic adenocarcinoma is ghrelin-responsive [55]. In addition, they have demonstrated that the PI3-K/Akt pathway is an important mediator of ghrelin’s stimulatory effects on migration and invasiveness in pancreatic adenocarcinoma cells. Proliferation was affected dose-dependently, being suppressed at higher ghrelin concentrations. D-Lys-GHRP6 (ghrelin’s antagonist) suppressed ghrelin-induced proliferation, invasion, and Akt phosphorylation [55]. Except from all these studies, which attest that ghrelin promote cell proliferation and differentiation in malignant tissues; there are a few others that investigated ghrelin’s anti-proliferation action and protection against cancer. More specifically, Volante et al. showed that ghrelin has antiproliferative actions [at relatively high concentrations (100 nmol/L to 1 mmol/L)] in thyroid carcinoma cell lines, suggesting that autocrine circuits may be operating in the growth control of follicular tumours. As the anaplastic thyroid carcinoma cell line ARO requires higher ghrelin concentrations for the inhibition of cell proliferation, compared to the papillary carcinoma-derived cell line, NPAP, it seems possible that cells that are less differentiated may express a lower-affinity or modified ghrelin-related receptor. Therefore, ghrelin may possibly act as a negative growth factor in thyroid carcinomas [47,48]. Moreover, in human lung carcinoma cell lines (CALU-1), Ghe et al. observed in vitro specific binding sites for GHSs, as their proliferation was inhibited by synthetic peptide analogues and analogues of hexareline, only while no inhibition was noted by synthetic no-peptidyl analogues (MK-0677) or ghrelin itself [49]. It was suggested that the inhibitory effect of the synthetic peptidyl GHSs on lung cancer cell lines was not a result of binding with GHS-R1a receptor, but another unknown up to that period receptor [49] e today it is believed to be GHS-R1b, as it was shown by other studies [56]. At last, Delhanty et al. proposed that ghrelin and its unacylated isoform stimulate the growth of adrenocortical tumour cells via an anti-apoptotic pathway [57]. To be more specific, the researchers presented a growth stimulatory effect of both acylated and unacylated ghrelin on the adrenocortical carcinoma cell line SW-13. The expression of preproghrelin mRNA and ghrelin protein by SW-13 cells suggests that ghrelin, and perhaps also unacylated ghrelin, may act as auto/paracrine factors in adrenocortical tumour growth, perhaps even tumourigenesis, and this is substantiated by the finding that [D-Lys3]GHRP6 antagonises not only ghrelin-stimulated, but also basal cell growth. The proliferative response to unacylated ghrelin suggests at least one new receptor-mediated signaling pathway in these cells. The absence of consistently expressed GHS-R1a and the ability of [D-Lys3]-GHRP6 to block both acylated and unacylated ghrelin effects suggest that this receptor could bind all three peptide ligands [57].

D. Nikolopoulos et al. Hence, it arises again the same question; is ghrelin carcinogenic or protective against malignancy? In fact, it is still not clear whether ghrelin boosts in the malignant transformation of benign cells. Rather bulk of reliable evidence shows that ghrelin promotes carcinogenesis instead of protecting it. The difference between these controversial studies is in the concentration of ghrelin used. For instance -as it has already mentioned- Jeffery, Waseem and Papotti [51,52,55,58] used the normal serum concentration of ghrelin in vitro (110e120 pM), whereas Cassoni and Ghe [49,50] used much more high concentration (1 mM), which is believed to be possibly cytotoxic. At last, there are many other researchers who investigated and observed the expression of ghrelin and GHS-R in carcinomas and in endocrine tumours, such as in the human thyroid carcinomas [47,48], in the pancreatic endocrine tumours (insoulinomas, gastrinomas, glucagonomas, VIPomas) [41,59,60], in the testicular tumours [61], in the astrocytomas [62e65]. But it has not yet proved if ghrelin’s administration promotes or inhibits carcinogenesis, so we cannot hypothesise any specific action for ghrelin in these tumours. We can only declare our strong belief that in the future ghrelin may even be used as a neoplastic prognostic factor or as a new therapeutic target. Therefore, we believe that it is very important to study ghrelin levels in gastric, intestinal and colorectal cancer as this peptide is mainly produced and secreted from the gastric and intestinal mucosa; and because there have been published lastly some very interesting studies that has to do with our request for ghrelin’s complementary activity in gastrointestinal malignancy.

Ghrelin and gastrointestinal tract cancer Nowadays there have been published a lot of studies that have proved immunoreactivity for ghrelin and its receptor in endocrine and non-endocrine tumours of the stomach and the intestine. The question that always arises, but it was not answered until the first semester of 2008 (Waseem et al., study), is if ghrelin axis contributes to GIT carcinogenesis or if it acts protectively against malignancy. Starting with the first study that was published in 2001 by Papotti et al., who detected ghrelin e at gene and protein levels e in the majority of gastric carcinoids [75% (IHC) and 81% (ISH)] and in a fraction of intestinal neuroendocrine tumours [27% (IHC), 72% (ISH) and 100% (RT-PCR)]. By using specific antibody for the ghrelin receptor, Papotti observed that GHS-R mRNA (types 1a and 1b) was absent in his study gastric carcinoids available for RT-PCR analysis, but was present in 63% of intestinal carcinoids. Whether or not negative cases truly lack the GHS-R or possess a different, currently unknown, receptor subtype was not cleared, but it is very possible [58]. Ghrelin-producing tumours should, therefore, be added to the list of hormone-producing gastrointestinal endocrine tumours, with special reference to gastric carcinoids. Enterochromaffin-like (ECL) cell carcinoids are the most common endocrine tumours of the corpus/fundus mucosa and are usually the consequence of hypergastrinemic conditions associated with chronic atrophic gastritis. The proportion of ghrelin-producing cells was much higher than

Ghrelin’s Role on Gastrointestinal Tract Cancer that in normal oxyntic mucosa [58,66]. Hypergastrinemic conditions leading to the proliferation of ECL and of the X/A cells could cause hyperplastic and neoplastic endocrine cell growth and induce consequently ghrelin overproduction by activating the ghrelin-producing cell compartment [56,67,68]. Although the whole spectrum of ghrelin functions is still incompletely defined, no reports exist in the literature on functioning gastric carcinoids having symptoms possibly related to GH release. One single case of multiple gastric carcinoid has been described until now; associated with a GH-producing pituitary adenoma, possibly related to hypergastrinemia-induced GHRH release [69]. By studying the circulating ghrelin levels in 78 patients with gastric or colorectal cancer, Huang et al. (2007) reported that there were no significant difference in plasma ghrelin levels among gastric cancer patients, colorectal cancer patients and control individuals. Furthermore, the plasma ghrelin levels were not influenced by tumour location or other hormone levels (GH, glucagon and cortisol levels) [70]. In another study with 80 gastric cancer patients, who underwent a radical subtotal or total gastrectomy, it was observed that the ghrelin levels in the gastric cancer tissues were significantly lower than those in the normal tissue and there was a significant difference according to the histological type of tumour. For instance, ghrelin levels were significantly lower in the differentiated tumour tissue than they were in the undifferentiated. Nevertheless, there was no significant difference in ghrelin levels regardless of the tumour stage, tumour depth of invasion, and lymph node metastasis [71]. These results suggest that the production of ghrelin in the gastric mucosa is influenced by gastric cancer and its differentiation. The significantly lower plasma ghrelin levels after gastrectomy indicate the stomach to be the main site of ghrelin production. In continuity to the above data, D’Onghia et al. observed, in a group of 29 colorectal cancer patients, that ghrelin levels were statistically lower in patients compared to healthy individuals, independent of tumour location [72]. Statistically significant differences in serum ghrelin levels between the patients and the control group were also found, with a significant fall from the tumour’s earlier stages (Dukes A or B1) to its later stages. This interesting result can probably be linked to the progressive loss of cell differentiation during the disease. Another intriguing finding is the negligible ghrelin expression in adenocarcinomas of oesophago-gastric tumours and the adjacent nonneoplastic mucosa. Mottershead et al. published the absence of ghrelin-producing cells in gastric and oesophageal adenocarcinomas whereas the highest level of ghrelin expression was noted in the non-neoplastic mucosa of the gastric corpus. As a result, it was hypothesised that the disruption of the gastric ghrelin-producing mechanism may occur during oesophago-gastric malignancy [73]. Likely, Asydin et al. were the first who observed ghrelin’s absence in a small number of patients suffering from gastric adenocarcinoma. This finding led the researchers to suggest that the ghrelin-producing capacity is possibly lost in a stage of the neoplasy [69]. Until now, the body of evidence suggests that ghrelin immunoreactive cells are present in the majority of the population of gastro-enteric tumours, and could therefore

e7 take part in gut endocrine growth, although ghrelinproducing tumours have not yet been reported to cause any endocrine effects in vivo. An intriguing recent finding is that of Waseem et al. who speculated that ghrelin can exert a proliferative activity in colorectal cancer cells. To be more specific, Waseem et al. have shown for the first time that colorectal cancer cells excessively secrete ghrelin in vitro and use it in autocrine and paracrine manner to promote their proliferative and invasive behaviour. Malignant colorectal tissue samples showed over-expression of ghrelin in a stage-dependent manner as opposed to normal colorectal tissue samples. The known functional receptor, GHS-R1a expression was dramatically lost in malignant colorectal cells and GHS-R1b expression was rather enhanced. This implies that ghrelin might be mediating its proliferative and invasive behaviour either through the ‘non-functional’ GHS-R1b, or through an unknown receptor subtype, which shares the same protein sequence with GHS-R1b. Furthermore, well-differentiated and moderately differentiated colorectal cancer samples present higher ghrelin expression as compared to controls [74]. The differentially enhanced co-expression of ghrelin and its receptors points toward the fact that ghrelin-GHS-R axis might be involved in malignant behaviour of colorectal carcinoma through autocrine/paracrine means. Normal human colonocytes produced minimal ghrelin, though target receptors in form of GHS-R1a and 1b existed in these cells. Probably this was the reason why normal colonocytes do not proliferate or invade independently in the absence of growth factors, as researchers speculated. However, well-differentiated (SW-48) and poorly differentiated (RKO) malignant cells, which produce more ghrelin, exhibited significant proliferative and invading/migrating action even in the absence of growth factor addition, in the culture medium (10% FBS). Lastly, this baseline proliferative and invasive/migratory pattern was almost completely abolished when researchers pre-treat the cells with either GHSR antagonist, D-[Lys3]-GHRP6 or a neutralising antibody specific to ghrelin. This implies that endogenous ghrelin is required for the proposed autocrine/paracrine stimulation of malignant intestinal epithelial cells to promote their baseline proliferation and invasion [74].

Conclusion In conclusion, ghrelin is a brain-gut peptide, which is mainly secreted by the stomach mucosa, but it is also expressed widely in a variety of tissues e in physiologic and neoplasmatic conditions e and, therefore, ghrelin may exert variable endocrine and paracrine effects. A specific modification in the peptide’s chain (acyliosis) is important for some of the biologic effects of that molecule, such as GH secretion, food intake and anabolic effects, whereas in other conditions, as for example in cell proliferation, that modification is not essential. An autocrine and/or paracrine function of ghrelin has recently been suggested in neoplasia and for a great number of tumours. Finally, immunopositivity for ghrelin and GHS-R has been identified in many endocrine and non-endocrine tumours of the gastrointestinal track, but many more studies have to be conducted, so that an accurate mechanism of ghrelin in physiological

e8 and pathophysiological conditions of gastrointestinal malignancy may be identified. An interesting newly published study demonstrated for the first time that ghrelin expression is enhanced in malignant colorectal cells and it promotes colorectal malignancy by advancing stage of tumour through an autocrine/paracrine mechanism. As a consequence, is ghrelin carcinogenic or protective against malignancy? In fact, still it is not clear whether ghrelin is truly carcinogenic or protective against malignancy. Rather bulk of reliable evidence shows that ghrelin promotes malignancy instead of protecting it. On the other hand, it is strongly believed that ghrelin will probably be a new therapeutic target for GIT cancer in the next decade, or a new prognostic factor, especially for end stage GI cancer.

Conflict of interest statement The authors declare that no one has conflicts of interests with the study presented.

Acknowledgments We thank Evita Alexopoulou for helping us with the editing of the review article.

References [1] Momany FA, Bowers CY, Reynolds GA, Chang D, Hong A, Newlander K. Design, synthesis, and biological activity of peptides which release growth hormone in vitro. Endocrinology 1981;108(1):31e9 [My paper]. [2] Bowers CY. Unnatural growth hormone-releasing peptide begets natural ghrelin. J Clin Endocrinol Metab 2001;86(4): 1464e9. [3] Rosicka M, Krsek M, Jarkovska Z, Marek J, Schreiber V. Ghrelin e a new endogenous growth hormone secretagogue. Physiol Res 2002;51:435e41. [4] DRCh Murray, Kamm MA, Bloom SR, Emmanuel AV. Ghrelin for the gasroenterologist: history and potential. Gastroenterology 2003;125:1492e502. [5] Kojima M, Hosoda H, Kangawa K. Ghrelin, a novel growthhormone-releasing and appetite-stimulating peptide from stomach. Best Pract Res Clin Endocrinol Metab 2004;18(4): 517e30. [6] Kojima M, Hosoda H, Matsuo H, Kangawa K. Ghrelin: discovery of the natural endogenous ligand for the growth hormone secretagogue receptor. Trends Endocrinol Metab 2001;3:118e22. [7] Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, et al. A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 1996;273: 974e7. [8] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402:656e60. [9] Korbonits M, Goldstone AP, Gueorguiev M, Grossman AB. Ghrelinda hormone with multiple functions. Front Neuroendocrinol 2004;25:27e68. [10] Arvat E, Broglio F, Aimaretti G, Benso A, Giordano R, Deghenghi R, et al. Ghrelin and synthetic GH secretagogues. Best Pract Res Clin Endocrinol Metab 2002;16(3):505e17. [11] Hosoda H, Kojima M, Matsuo H, Kangawa K. Ghrelin and desacyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue. Biochem Biophys Res Commun 2000; 279:909e13.

D. Nikolopoulos et al. [12] Hosoda H, Kojima M, Mizushima T, Shimizu S, Kangawa K. Structural divergence of human ghrelin. Identification of multiple ghrelin-derived molecules produced by posttranslational processing. J Biol Chem 2003;278:64e70. [13] Banks WA, Tschop M, Robinson SM, Heiman ML. Extent and direction of Ghrelin transport across the bloodebrain barrier is determined by its unique primary structure. J Pharmacol Exp Ther 2002;302:822e7. [14] Bednarek MA, Feighner SD, Pong SS, McKee KK, Hreniuk DL, Silva MV, et al. Structure and function studies on the new growth hormone releasing peptide, ghrelin: minimal sequence of Ghrelin necessary for activation of growth hormone secretagogue receptor 1a. J Med Chem 2000;43:4370e6. [15] Broglio F, Benso A, Gottero C, Prodam F, Gauna C, Filtri L, et al. Non-acylated ghrelin does not possess the pituitaric and pancreatic endocrine activity of acylated ghrelin in humans. J Endocrinol Invest 2003;26:192e6. [16] Bedendi I, Alloatti G, Marcantoni A, Malan D, Catapano F, Ghe ´ C, et al. Cardiac effects of ghrelin and its endogenous derivatives des-octanoyl ghrelin and des-Gln(14)-ghrelin. Eur J Pharmacol 2003;476:87e95. [17] Cassoni P, Papotti M, Ghe C, Catapano F, Sapino A, Graziani A, et al. Identification, characterization, and biological activity of specific receptors for natural (ghrelin) and synthetic growth hormone secretagogues and analogs in human breast carcinomas and cell lines. J Clin Endocrinol Metab 2001;86: 1738e45. [18] Thompson NM, Gill DA, Davies R, Loveridge N, Houston PA, Robinson ICAF, et al. Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mechanism independent of GHS-R1a. Endocrinology 2004;145:232e42. [19] Jeffery PL, Herington AC, Chopin LK. The potential autocrine/paracrine roles of ghrelin and its receptor in hormonedependent cancer. Cytok Growth Factor Rev 2003;14:113e22. [20] Wang HJ, Geller F, Dempfle A, Scha ¨uble N, Friedel S, Lichtner P, et al. Ghrelin receptor gene: identification of several sequence variants in extremely obese children and adolescents, healthy normal-weight and underweight students, and children with short normal stature. J Clin Endocrinol Metab 2004;89:157e62. [21] Date Y, Kojima M, Hosoda H, Sawaguchi A, Mondal MS, Suganuma T, et al. Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 2000;141:4255e61. [22] Dornonville dlC, Bjorkqvist M, Sandvik AK, Bakke I, Zhao CM, Chen D, et al. A-like cells in the rat stomach contain ghrelin and do not operate under gastrin control. Regul Pept 2001;99: 141e50. [23] Rindi G, Necchi V, Savioet A, Zoli M, Locatelli V, Raimondo F, et al. Characterisation of gastric ghrelin cells in man and other mammals: studies in adult and fetal tissues. Histochem Cell Biol 2002;21:17e24. [24] Kojima M, Kangawa K. Ghrelin, an orexigenic signalling molecule from the gastrointestinal tract. Curr Opin Pharmacol 2002;2:665e8. [25] Ariyasu H, Takaya K, Tagami T, Yasar A, Colakoglu N, Kelestimur H. Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans. J Clin Endocrinol Metab 2001;86: 4753e8. [26] Krsek M, Rosicka M, Haluzik M, Svobodova ´ J, Kotrlı´kova ´ E, Justova ´ V, et al. Plasma ghrelin levels in patients with short bowel syndrome. Endocr Res 2002;28:27e33. [27] Sakata I, Nakamura K, Yamazaki M, Matsubara M, Hayashi Y, Kangawa K, et al. Ghrelin-producing cells exist as two types of cells, closed- and opened-type cells, in the rat gastrointestinal tract. Peptides 2002;23:531e6.

Ghrelin’s Role on Gastrointestinal Tract Cancer [28] Leonetti F, Silecchia G, Iacobellis G, Ribaudo MC, Zappaterreno A, Tiberti C, et al. Different plasma ghrelin levels after laparoscopic gastric bypass and adjustable gastric banding in morbid obese subjects. J Clin Endocrinol Metab 2003;88:4227e31. [29] Cowley MA, Smith RG, Diano S, Tscho ¨p M, Pronchuk N, Grove KL, et al. The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 2003;37:649e61. [30] Korbonits M, Bustin SA, Kojima M, Jordan S, Adams EF, Lowe DG, et al. The expression of the growth hormone secretagogue receptor ligand ghrelin in normal and abnormal human pituitary and other neuroendocrine tumors. J Clin Endocrinol Metab 2001;86:881e7. [31] Muccioli G, Ghe C, Ghigo MC, Papotti M, Arvat E, Boghen MF, et al. Specific receptors for synthetic GH secretagogues in the human brain and pituitary gland. J Endocrinol 1998;157: 99e106. [32] Muccioli G, Papotti M, Locatelli V, Ghigo E, Deghenghi R. Binding of 125I-labeled ghrelin to membranes from human hypothalamus and pituitary gland. J Endocrinol Invest 2001; 24:RC7e9. [33] Dixit VM, Schaffer EM, Taub DD. Ghrelin is expressed and secreted from human T lymphocytes and inhibits activation and leptin induced inflammatory cytokine expression via functional, lipid raft-associated growth hormone secretagogue receptors (GHS-R): Proceedings of the 85th Annual Meeting of the Endocrine Society 2003; OR51eOR53. [34] Hattori N, Saito T, Yagyu T, Jiang BH, Kitagawa K, Inagaki C. GH, GH receptor, GH secretagogue receptor, and ghrelin expression in human T cells, B cells, and neutrophils. J Clin Endocrinol Metab 2001;86:4284e91. [35] Volante M, Fulcheri E, Allia E, Cerrato M, Pucci A, Papotti M. Ghrelin expression in fetal, infant, and adult human lung. J Histochem Cytochem 2002;50:1013e21. [36] Gualillo O, Caminos JE, Blanco M, Garcı`a-Caballero T, Kojima M, Kangawa K, et al. Ghrelin, a novel placentalderived hormone. Endocrinology 2001;142:788e94. [37] Caminos JE, Tena-Sempere M, Gaytan F, Sanchez-Criado JE, Barreiro ML, Nogueiras R, et al. Expression of ghrelin in the cyclic and pregnant rat ovary. Endocrinology 2003;144: 1594e602. [38] Gaytan F, Barreiro ML, Chopin LK, Herington AC, Morales C, Pinilla L, et al. Immunolocalization of ghrelin and its functional receptor, the type 1a growth hormone secretagogue receptor in the cyclic human ovary. J Clin Endocrinol Metab 2003;88:879e87. [39] Barreiro ML, Gaytan F, Caminos JE, Pinilla L, Casanueva FF, Aguilar E, et al. Cellular location and hormonal regulation of ghrelin expression in rat testis. Biol Reprod 2002;67:1768e76. [40] Tena-Sempere M, Barreiro ML, Gonzalez LC, Gayta ´n F, Zhang FP, Caminos JE, et al. Novel expression and functional role of ghrelin in rat testis. Endocrinology 2002;143:717e25. [41] Volante M, Allia E, Gugliotta P, Funaro A, Broglio F, Deghenghi R, et al. Expression of ghrelin and of the GH secretagogue receptor by pancreatic islet cells and related endocrine tumors. J Clin Endocrinol Metab 2002;87:1300e8. [42] Date Y, Nakazato M, Hashiguchi S, Dezaki K, Mondal MS, Hosoda H, et al. Ghrelin is present in pancreatic alpha-cells of humans and rats and stimulates insulin secretion. Diabetes 2002;51:124e9. [43] Prado CL, Pugh-Bernard AE, Elghazi L, Sosa-Pineda B, Sussel L. Ghrelin cells replace insulin-producing {beta} cells in two mouse models of pancreas development. Proc Natl Acad Sci USA 2004;101:2924e9. [44] Wierup N, Svensson H, Mulder H, Sundler F. The ghrelin cell: a novel developmentally regulated islet cell in the human pancreas. Regul Pept 2002;107:63e9.

e9 [45] Kim K, Arai K, Sannot N, Osamura RY, Teramoto A, Shibasaki T. Ghrelin and GHSR mRNA expression in human pituitary adenomas. Clin Endocrin 2001;54:759e68. [46] Nanzer AM, Khalaf S, Mozid AM, Fowkes RC, Patel MV, Burrin JM, et al. Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway. Eur J Endocrinol 2004;151:233e40. ` C, Deghenghi R, [47] Cassoni P, Papotti M, Catapano F, Ghe Ghigo E, et al. Specific binding sites for synthetic growth hormone secretagogues in non-tumoral and neoplastic human thyroid tissue. J Endocrinol 2000;165:139e46. [48] Volante M, Allia E, Fulcheri E, Cassoni P, Ghigo E, Muccioli G, et al. Ghrelin in fetal thyroid and follicular tumors and cell lines. Am J Pathol 2003;162(2):645e54. [49] Ghe C, Cassoni P, Catapano F, Marrocco T, Deghenghi R, Ghigo E, et al. The antiproliferative effect of synthetic peptidyl GH secretagogues in human CALU-1 lung carcinoma cells. Endocrinology 2002;143:484e91. [50] Cassoni P, Ghe C, Marrocco T, Tarabra E, Allia E, Catapano F, et al. Expression of ghrelin and biological activity of specific receptors for ghrelin and des-acyl ghrelin in human prostate neoplasms and related cell lines. Eur J Endocrin 2004;150: 173e84. [51] Jeffery PL, Murray RE, Yeh AH, McNamara JF, Duncan RP, Francis GD, et al. Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines. Endocr Relat Cancer 2005;12: 839e50. [52] Jeffery PL, Herington AC, Chopin LK. Expression and action of the growth hormone releasing peptide ghrelin and its receptor in prostate cancer cell lines. J Endocrinol 2002;172:R7e11. [53] Yeh AH, Jeffery PL, Duncan RP, Herington AC, Chopin LK. Ghrelin and a novel preproghrelin isoform are highly expressed in prostate cancer and ghrelin activates mitogenactivated protein kinase in prostate cancer. Clin Cancer Res 2005;11(23):8295e303. [54] Murata M, Okimura Y, Iida K, Matsumoto M, Sowa H, Kaji H, et al. Ghrelin modulates the downstream of insulin signaling in hepatoma cells. J Biol Chem 2002;277:5667e74. [55] Duxbury MS, Waseem T, Ito H, Robinson MK, Zinner MJ, Ashley SW, et al. Ghrelin promotes pancreatic adenocarcinoma cellular proliferation and invasiveness. Biochem Biophys Res Commun 2003;309:464e8. [56] Arnaldi G, Mancini T, Kola B, Appolloni G, Freddi S, Concettoni C, et al. Cyclical Cushing’s syndrome in a patient with a bronchial neuroendocrine tumor (typical carcinoid) expressing ghrelin and growth hormone secretagogue receptors. J Clin Endocrinol Metab 2003;88(12):5834e40. [57] Delhanty PJD, van Koetsveld PM, Gauna C, van de Zande B, Vitale G, Hofland LJ, et al. Ghrelin and its unacylated isoform stimulate the growth of adrenocortical tumor cells via an antiapoptotic pathway. Am J Physiol Endocrinol Metab 2007;293: E302e9. [58] Papotti M, Cassoni P, Volante M, Deghenghi R, Muccioli G, Ghigo E. Ghrelin-producing endocrine tumors of the stomach and intestine. J Clin Endocrinol Metab 2001;86:5052e9. [59] Iwakura H, Hosoda K, Doi R, Komoto I, Nishimura H, Son C, et al. Ghrelin expression in islet cell tumors: augmented expression of ghrelin in a case of glucagonoma with multiple endocrine neoplasm type I. J Clin Endocrinol Metab 2002;87: 4885e8. [60] Ekeblad S, Lejonklou MH, Grimfjard P, Johansson T, Eriksoon B, Grimelius L, et al. Co-expression of ghrelin and its receptor in pancreatic endocrine tumours. Clin Endocrinol 2007;66:115e22. [61] Gaytan F, Barreiro ML, Caminos JE, Chopin LK, Herington AC, Morales C, et al. Expression of ghrelin and its functional receptor, the type 1a growth hormone secretagogue receptor,

e10

[62]

[63]

[64] [65] [66]

[67]

[68]

D. Nikolopoulos et al. in normal human testis and testicular tumors. J Clin Endocrinol Metab 2004;89:400e9. Dixit VD, Weeraratna AT, Yang H, Bertak D, Cooper-Jenkins A, Riggins GJ, et al. Ghrelin and the growth hormone secretagogue receptor constitute a novel autocrine pathway in astrocytoma motility. J Biol Chem 2006;281(24):16681e90. Puduvalli VK, Kyritsis AP, Hess KR, Bondy ML, Fuller GN, Kouraklis GP, et al. Patterns of expression of Rb and p16 in astrocytic gliomas, and correlation with survival. Int J Oncol 2000;17(5):963e9. de Herder WW. Biochemistry of neuroendocrine tumours. Best Pract Res Clin Endocrinol Metab 2007;21(1):33e41. Leontiou CA, Franchi G, Korbonits M. Ghrelin in neuroendocrine organs and tumours. Pituitary 2007;10:213e25. Kojima K, Miyake M, Nakagawa H, Yunoki Y, Ogurusu K, Saino S, et al. Multiple gastric carcinoids and pituitary adenoma in type A gastritis. Intern Med 1997;36:787e9. Rindi G, Azzoni C, La Rosa S, Klersy C, Paolotti D, Rappel S, et al. ECL cell tumor and poorly differentiated endocrine carcinoma of the stomach: prognostic evaluation by pathological analysis. Gastroenterology 1999;116:532e42. Tsolakis A, Portella-Gomes GM, Stridsberg M, Grimelius L, Sundin A, Eriksson BK, et al. Malignant gastric ghrelinoma with hyperghrelinemia. J Clin Endocrinol Metab 2004;89(8):3739e44.

[69] Aydin S, Ozercan IH, Dagli F, Aydin S, Dogru O, Celebi S, et al. Ghrelin immunohistochemistry of gastric adenocarcinoma and mucoepidermoid carcinoma of salivary gland. Biotech Histochem 2005;80(3-4):163e8. [70] Huang Q, Fan Y-Z, Ge B-J, Zhu Q, Tu Z-Y. Circulating ghrelin in patients with gastric or colorectal cancer. Dig Dis Sci 2007;52: 803e9. [71] Ji YA, Min-Gew Ch, Jae HN, Sohn TS, Jin DK, Kim S. Clinical significance of ghrelin concentration of plasma and tumor tissue in patients with gastric cancer. J Surg Res 2007;143: 344e9. [72] D’Onghia V, Leoncini R, Carli R, Santoro A, Giglioni S, Sorbellini F, et al. Circulating gastrin and ghrelin levels in patients with colorectal cancer: correlation with tumour stage, Helicobacter pylori infection and BMI. Biomed Pharmacother 2007;61:137e41. [73] Mottershead M, Karteris E, Barclay JY, Suortamo S, Newbold M, Randeva H, et al. Immunohistochemical and quantitative mRNA assessment of ghrelin expression in gastric and oesophageal adenocarcinomas. J Clin Pathol 2007;60: 405e9. [74] Waseem T, Javaid-ur- Rehman, Ahmad F, Azam M, Qureshi AM. Role of ghrelin axis in colorectal cancer: a novel association. Peptides 2008;29:1369e76.