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Melatonin  receptor  expression  in  the  normal  human  gastrointestinal  tract  and  in  gastrointestinal  neuroendocrine  tumors

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Melatonin  receptor  expression  in  the  

normal  human  gastrointestinal  tract  and  

in  gastrointestinal  neuroendocrine  

tumors  

 

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Abstract  

Söderquist, F. Melatonin receptor expression in the normal human gastrointestinal tract and in gastrointestinal neuroendocrine tumors

Melatonin is present in peripheral tissues and regulates many functions. The largest source, according to animal studies, is the gastrointestinal tract (GIT). Melatonin receptors, MT1 and MT2 have been identified in rat intestine, but in humans receptor expression has not been thoroughly characterized.

This study aims to map the expression of melatonin and its two receptors both in normal human GIT as well as in small intestine neuroendocrine tumors (SI-NETs) derived from enterochromaffin cells.

Biopsies from normal GIT from 21 individuals and from SI-NETs from 39 patients were immunohistochemically stained for melatonin, MT1 and MT2. Melatonin levels in plasma from 20 patients with SI-NETs were measured.

Melatonin stainings were not complete due to methodological problems. Positive expression of both MT1 and MT2 was found in the epithelium of the normal GIT, in sections representing stomach (n=5), small intestine (n=4) and large intestine (n=12). Positive expression was also found for MT2 in endocrine cells in crypts throughout the GIT. In the tumor sections, positive expression for MT1 was rare, while the majority of sections studied were positive for MT2. The intensity of the staining was not related to clinical parameters but MT2 expression was stronger in primary tumors than in metastases (p=0,01).

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Sammanfattning  på  svenska

 

Söderquist, F. Uttrycket av melatoninreceptorer i normal tarm hos människa och i gastrointestinala neuroendokrina tumörer

Melatonin, ett hormon från tallkottkörteln som styr sömn och dygnsrytm finns även i perifera vävnader och reglerar många andra funktioner. Djurstudier har visat att den största källan till melatonin är mag- tarmkanalen. Melatoninreceptorerna , MT1 och MT2 har identifierats i tarmen hos råtta, men uttrycket i tarm hos människa är inte lika noggrannt studerat.

Syftet med den här studien var att kartlägga uttrycket av melatonin och dess båda receptorer både i normal tarm hos människa samt i neuroendokrina tumörer (tunntarmskarcinoider) utgående från enterokromaffinceller.

Biopsier från normal tarm från 21 individer och från karcinoider från 39 patienter färgades med immunohistokemisk teknik för MT1 och MT2. Plasmanivåer av melatonin mättes hos 20 patienter med tunntarmskarcinoider.

Färgningar för melatonin är inte kompletta på grund av metodproblem. Positivt uttryck för både MT1 och MT2 identifierades i epitelet av normal tarm, i snitt från magsäck (n=5), tunntarm (n=4) och tjocktarm (n=12). För MT2 fanns positivt uttryck även i endokrina celler i tarmen. I tumörerna var endast ett fåtal snitt positva för MT1, medan majoriteten var positiva för MT2. Intensiteten av uttrycket var inte relaterad till kliniska parametrar, men för MT2-receptorn var färgningen starkare i primärtumör än i metastas (p=0,01).

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Introduction

Melatonin is an indolamine derived from tryptophan, two enzymatic steps from serotonin [1]. It is mainly known as a pineal gland neuroendocrine hormone that regulates sleep and circadian rhythm. However, melatonin is also present in peripheral tissue and produced in organs such as retina, gastrointestinal tract (GIT), bone marrow, lymphocytes, skin [2-4] and secreted in breast milk [5] saliva [6] and bile [7].

Surprisingly, animal studies indicate that the largest source of melatonin is the gastrointestinal mucosa and when compared to the production in the pineal gland, the total amount of gastrointestinal melatonin is estimated to be 400 times higher [8]. The amount of melatonin also correlates to the number of enterochromaffin cells (EC-cells) in the mucosa [9]. Levels of melatonin vary in relation to fasting and food intake. In pinealectomized rats, melatonin levels in the portal vein increase after tryptophan administration [10]. In pigs, levels of melatonin do not follow a circadian rhythm but are elevated after food intake in blood from the portal vein [11]. In mice, short term fasting (24h and 48 h) resulted in increased levels of melatonin in the GIT, particularly in the stomach [12]. In humans, melatonin levels in saliva are elevated after a meal [13], while short term fasting (2 days) reduces nocturnal concentrations of melatonin in serum [14].

There are two known receptors for melatonin, melatonin receptor type 1A (MT1) and melatonin receptor type 1B (MT2). They are both G-protein coupled and act by affecting intracellular messengers such as cAMP, cGMP och Ca2+ [15]. Table 1 summarizes the current knowledge of melatonin receptor expression in humans.

Table 1. Localization of melatonin receptor in human tissue and methods used to identify them. References indicated.

Localization   Receptor   Methods  used   Reference  

SCN   MT1   PCR,  mRNA   [16]  

Hippocampus   MT1,  MT2   IHC   [17,  18]  

Cerebellum   MT1,  MT2   mRNA,  in  situ  hybridization   [19]  

Retina   MT1,  MT2   PCR,  for  MT1  IHC   [20]  

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(monocytes,  B  and  T   lymphocytes  and   Natural  Killer  cells)  

lymphoid  cell  lines)    

Salivary  glands   MT1   IHC   [23]  

Gallbladder   MT1   PCR,  western  blot,  IF   [24]  

Gastrointestinal   tract,  duodenum  

MT2     Inhibitor  test   [25,  26]  

Gastrointestinal   tract,  colon  

MT1   PCR  mRNA,  western  blot,   IHC   [27]   Pancreas   MT1(alpha-­‐cells),  MT2   (beta-­‐cells)   mRNA,  IHC   [28-­‐30]   Heart,  coronary   arteries,  aorta   MT1,  MT2   PCR   [31,  32]   Breast   MT1   IHC   [33,  34]  

Ovaries   MT1,  MT2   PCR  mRNA,  southern  blot   [35,  36]   Prostate   Functional  binding  sites  

MT1  in  cancer  tissues  

Autoradiography   IHC    

[37]   [38]  

Adipose  tissue   MT1,  MT2   PCR  mRNA   [39]  

Skin   MT1,  (MT2)   PCR   [40]  

Abbreviations: PCR= Polymerase chain reaction, IHC= Immunohistochemistry, IF= Immunoflouroscence

MRNA transcripts for both receptors have been detected in many different tissues in humans [41, 42], while protein expression is not studied as extensively. A study by Nemeth et al. has demonstrated the expression of MT1 in human colon using immunohistochemistry [27]. Another study by Sjöblom et al. investigated the effects of melatonin on intracellular calcium (Ca2+) in both rat and human duodenum. The

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primarily in the muscularis mucosae and muscularis externa[46]. Protein expression of both melatonin receptors in human GIT has, to our knowledge, not been systematically characterized.

Melatonin can also bind to nuclear receptors; the retinoid related orphan nuclear hormone receptor (RZR/RORalfa), subtypes RZRα, RORα, RORα2 and RZRβ [47]. Subtypes of the nuclear receptor family display tissue specificity but their function is largely unknown [48].

Melatonin is a small lipophilic compound that diffuses easily through membranes and quickly accesses different tissues. It can act in an endocrine, paracrine or autocrine fashion. Besides acting as a regulator of circadian rhythm, melatonin has been shown to have many other functions. In the immune system, melatonin acts as an immunomodulator by binding to its specific receptors. The effects seem to be both stimulatory and inhibitory [49-51]. Melatonin is also a powerful antioxidant, acting as a scavenger of free radicals, directly neutralizing several toxic oxygen- and nitrogen-based reactants in a non-receptor-dependent manner [52]. These anti-inflammatory effects of melatonin have been described in many studies [53, 54].

Melatonin has been shown to promote cell survival in normal tissues [55-57], but to have oncostatic effects in various types of cancer [58-61]. For example melatonin induced caspase activity and apoptosis in human malignant lymphoid cell lines [22]. In prostate cancer, receptor dependent anti-proliferative actions have been shown via up-regulation of p27kip1. Melatonin binds to MT1 and via PKA and PKC constitutively active NF-kB is inhibited, releasing the inhibition of the p27kip1 promoter resulting in up-regulation of gene- and protein expression [62].

Melatonin affects insulin and glucagon secretion [63] presumably via MT1 and MT2 receptor subtypes present in human pancreas. MT2 is primarily localized in the beta-cells [28], while MT1 has been identified in alpha-cells and to a lesser degree in beta-cells [30]. There is an association between decreased melatonin secretion and increased risk of development of type 2 diabetes [64] and rare variants in the gene encoding the MT2-receptor subtype, that lead to impaired melatonin signaling increase the risk for type 2 diabetes [65]. Melatonin even may influence other hormones regulating hunger and satiety such as leptin and ghrelin [66, 67].

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gastrointestinal tract [69].  In blood vessels, melatonin activation of the MT2 receptor causes vasodilation while MT1 mediates vasoconstriction [70].

Melatonin also exhibits significant protective effects against damage to the intestinal mucosa. Luminal melatonin increases bicarbonate secretion in the duodenum in response to acidic luminal contents via MT2 [25]. In rats, melatonin also reduces epithelial paracellular permeability preventing substances such as endotoxins from leaking in causing inflammation [71], in addition to its previously mentioned anti-inflammatory effects by acting as a scavenger of free radicals [52].

The EC-cells are neuroendocrine cells scattered along the gastrointestinal tract that are known to produce and secrete serotonin. The EC-cells have also been implied to be the source of gastrointestinal melatonin [8]. Tumors derived from EC-cells, small intestine neuroendocrine tumors (SI-NETs), traditionally called midgut carcinoids are also known to produce serotonin. SI-NETs are usually tumors with long survival expectancy although poorer prognosis can be predicted in cases where proliferation index Ki67 is ≥1% and the tumor has a solid growth pattern [72]. Whether or not the tumors also produce melatonin has yet to be investigated.

Objectives  

We hypothesize that melatonin is produced in serotonin producing enterochromaffin cells (EC-cells) in humans with the primary location in the ileum and that the tumors derived from EC-cells, SI-NETs also produce melatonin.

We further hypothesize that the receptors for melatonin (MT1 and MT2) are expressed in normal human intestinal mucosa as well as in SI-NETs.

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Materials  and  methods  

Patient  samples  

To study the expression of melatonin and its receptors in the normal gastrointestinal tract, biopsies obtained from patients who underwent surgery for other reasons were used. In total 21 patients (17 purchased from Asterand, USA and 4 from the Department of Pathology, Uppsala University Hospital). Clinical data for these patients were unknown.

Clinical records were collected for 59 patients with SI-NETs, 30 women and 29 men, diagnosed at the Laboratory of Pathology and Cytology and treated at the Department of Endocrine Oncology, Uppsala University Hospital, Sweden. Tumor biopsies were available from 39 of the 59 patients and plasma samples from the remaining 20 patients.

For 28 of the 39 patients where tumor biopsies were obtained, sections from both the primary tumor and metastasis were available and for the remaining only one or the other. Only sections where both primary tumor and metastasis could be evaluated were included in statistical analyses comparing the two.

For patients marked as deceased in the medical journal system Cambio Cosmic, patient survival analysis was calculated from time of diagnosis to death. The remaining patients were considered to still be alive and survival was calculated from time of diagnosis until present date (June1st2013). For international patients referred to the Department of Endocrine Oncology in Uppsala, follow-up could not be traced and they were censored in statistical analyses.

Information extracted from the medical records included Body Mass Index (BMI), systolic blood pressure, smoking history, diagnosis of diabetes and use of antidepressant or anti-anxiety drugs as well as medication for sleep disorders. Smoking history was classified as current, past or none. The parameters documented were those from registration before surgery or from time of blood sampling in the group of patients where no biopsies were obtained. Plasma samples were collected after an overnight fast at two occasions, before start of interferon treatment and after. Plasma levels of melatonin were measured as well as the tumor marker Chromogranin A (CgA) and the serotonin metabolite Urinary 5-hydroxyindoleacetic acid ( U-5HIAA).

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Hostpital (Dnr: 2007/143 and 2007/143/1 GK 2012-09-14) and all included patients signed an informed consent.

Immunohistochemistry  and  microscopial  assessments  

Samples were fixed in 10 % buffered neutral formalin and routinely processed to paraffin. Four µm thin sections were cut and attached to glass slides. Sections were deparaffinized with xylene and rehydrated to distilled water with descending concentrations of ethanol. Immunohistochemical staining for melatonin, MT1 and MT2 was performed using either the streptavidin-biotin complex technique or the DAKO EnVision system according to the manufacturer’s instructions. Diaminobenzidine was used as chromogen. For visualization of nuclei the sections were counterstained with Mayer’s haematoxylin. Staining for serotonin was also performed. For primary antibodies used for immunohistochemistry see Table 2.

Table 2. Antibodies used in the immunohistochemistry application.

Antibody  target   Company   Dilution  

Melatonin  Polyclonal     AB-­‐T177  

Advanced   Targeting   Systems,   San   Diego,  CA,  USA  

1/500    

Anti-­‐MTR-­‐1A   ACC-­‐250761  

Abbiotec,  San  Diego,  USA   1/100  

Anti-­‐Melatonin  Receptor  1B   ABIN122307  

DAKO,   Sweden   AB,   Stockholm,   Sweden  

1/100  

Serotonin   Clone  5HT-­‐H209  

DAKO,   Sweden   AB,   Stockholm,   Sweden  

1/100  

Primary antibodies against MTR-1A and MTR-1B were blocked using blocking peptides. For MTR-1A, a peptide from another company and antibody was used because the corresponding peptide was nor commercially available. Control staining without the primary antibody for melatonin was performed. Dilution series were performed to verify that the concentration of the primary antibody truly had an effect on the result and that the same pattern was seen with only variations in intensity.

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intestine (n=4) and large intestine (n=12). Tumor sections consisted of primary tumor (n=30) and metastases (n=36). All specimens were coded and examined microscopically by two observers, using both low (100x) and high magnification (400x). Sections from normal tissue were classified as positive or negative for MT1 and MT2 and localization was documented. All tumor sections were subjectively assessed regarding the positive and negative expression for melatonin, MT1 and MT2 as well as the intensity of the staining when positive. The sections were classified using a scoring system where zero (0) represented negative, (1) weakly positive (2) moderately positive and (3) strongly positive.

Positive control sections from tissues known to express MT1 and MT2 (skin, pancreas) were used for comparison. Negative outcomes in tumor sections were assessed as negative only if staining was present in control cells (epithelium, immune cells).

Plasma  analyses  

Plasma levels of melatonin were measured using competitive enzyme-linked immunosorbent assay (ELISA) kits (MELATONIN ELISA EK-DSM Bühlmann, Skafte MedLab, Odensala, Sweden) and results were registered using the instrument Wallac Victor2™.

Statistical  analyses  

Data was stored in an Excel database and analyzed using the statistical program package SPSS 21.0 (SPSS Inc., Chicago, IL, USA). Statistical analyses were conducted separately for the two groups of patients; those where histopathological specimens were available and those where plasma samples were collected. Patient characteristics was presented in descriptive statistical analyzes. To look for correlations between receptor expression or plasma levels of melatonin and clinical parameters, Pearson’s correlation or Mann-Whitney Test was used.

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Results

 

Antibody  specificity  

Tissues known to express melatonin receptor MT1 and MT2, skin [40] and pancreas [30], were used as positive controls and included in each batch of stainings (Fig. 1).

For the MT1 receptor blocking, the blocking peptide was from another company and not perfectly matched to the antibody and the blocking was partial. Control staining without the primary antibody for melatonin showed background activity. This background activity was completely removed by adding a biotin/avidin blocking step to the protocol and staining for melatonin is underway but not yet complete.

Staining for MT2 was negative for internal positive controls in 2 sections from normal tissue (1 from small intestine and 1 from large intestine) and for 10 cases in tumor section(6 from primary tumor and 4 from metastasis). They were redone, but could not be evaluated within the time frame of this study and were excluded from statistical analyzes.

Fig. 1: Antibody specificity for MT2 in control specimen of human skin, magnification 100x. Positive immunohistochemical expression in epidermis a), expression blocked by blocking peptide b).

Expression  of  MT1  and  MT2  in  normal  GIT  

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crypts where positive staining could be seen. Single cells with endocrine appearance were weakly positive in one of the sections. In vascular endothelium variations in the positive expression for MT1 could be seen.

The expression of MT2 was most prominent in the epithelium, localization was predominant in the nuclei. Positive staining was present in muscle tissue, also located to the cell nuclei. Cells with endocrine appearance in the deep of the crypts were positive in 11 of 20 sections (55 %) and all of the sections from the ileum showed staining of cells with endocrine appearance. The expression was in the cytoplasm in contrast to the epithelial cells. These cells were lower in numbers when compared to serotonin producing cells. Receptor expression in tumor section is presented in Fig. 2 and 3.

  Fig 2. Expression of melatonin receptors MT1 and MT2 in different segments of the normal gastrointestinal tract.

 

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Fig. 3: Immunohistichemical staining for melatonin receptors; perinuclear expression of MT1 in epithelial cells of human colonic mucosa A), nuclear expression of MT2 in epithelial cells of human colonic mucosa B) and expression of MT2 in cells with endocrine appearance in crypts of ileum C).  

 

Expression  of  MT1  and  MT2  in  SI-­‐NETs  

In the group of patients where tumor biopsies were available, totally 39 patients, median age was 61 years [40-85] for detailed clinical data see Table 3. In 32 cases (82.1 %) liver metastases were present at the time of operation and 17 (43.6 %) had more than five metastases and were classified as massive disease. All patients where data was obtainable had lymph node metastases. At the time of operation, 11 patients used antidepressants, 8 used sleeping pills and 4 used drugs for anxiety.

Table 3. Patient characteristics for the immunohistochemistry study.

 

Number  of  patients     39  

Age  at  operation  (median  years)     61  [40-­‐85]  

Sex     Male   19  

  Female   20  

Treatment  before  operation   No  Treatment   16  

  SOM   18  

  INF   17  

  SOM  and  INF   12  

*Survival  (median  months)     92  [6-­‐448]  

Status   Dead  with  disease   16  

  Alive  with  disease   18  

  Lost  to  follow-­‐up   5  

BMI  (median  kg/m2)     24.4  [14.9-­‐32.7]  

Systolic   blood   pressure   (median   mmHg)  

  140  [110-­‐180]  

Smoking  history  (n)   Current   4  

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  No   24   Antidepressants  (n)     11   Anti-­‐anxiety  medication  (n)     4   Sleeping  pills  (n)     8   Diabetes  (n)     5   Ki67  (median  %)     0.5  [0-­‐8]  

U-­‐5HIAA  (median  μmol/24h)       158  [10-­‐1386]  

CgA  (median  nmol/L)     16  [3-­‐355]  

*Survival is calculated as months from diagnosis to death In one case no documentation of smoking history was found

U-5HIAA: Urinary 5-hydroxyindoleacetic acid (serotonin metabolite) normal reference is 50 µmol/24h;

CgA: Chromogranin A, normal reference value is <4nmol/L;

Ki67 (%), proliferation index in tumor areas with highest proliferation.

Abbreviations: SOM= Somatostatin analogue treatment, INF= interferon treatment  

The expression of the MT1 receptor was negative in 55 of 69 sections (80%) and weakly positive in 14 of 69 sections (20%), with areas of weak staining in the tumor cells. Generally there was a stronger intensity towards the epithelial border and in the borders around groupings of tumor cell clusters. Positive expression could be seen in the brush border of the epithelium, which served as an internal positive control .

In the 57 sections assessed for MT2, 56 (98%) were positive and divided as weakly (32%), moderately (47%) or strongly (19%) positive. Tumor specimens stained for MT2 was categorized into two outcomes: scores low expression (negative or weakly positive) and high expression (moderately or strongly positive) and sections of primary tumor was compared to metastasis using Fisher’s exact test. Higher frequency of strong expression was found in primary tumor (p=0,0138). Positive expression for MT1 and MT2 in tumor sections is presented in Fig. 4 and 5.

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  Fig. 4. Expression of the MT1 and MT2 receptor in primary tumor and metastasis.  

 

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  Fig. 5. Representative cases of tumor sections immunostained for the MT2 receptor. In a negative A), weakly positive B), moderately positive C) and strongly positive neuroendocrine tumor D).

 

Analyses  of  plasma  samples  from  patients  with  SI-­‐NETs  before  and  after  interferon   treatment  

The group where plasma samples were available consisted of 20 patients, 11 women and 9 men. At the baseline time of plasma collection 18 (90 %) of the patients had lymph node metastases and 1 had liver metastases: for detailed results see Table 4. Of the 20 patients, 3 used antidepressants, 3 used anti-anxiety medication and 3 used sleeping pills.

Table 4. Patient characteristics for plasma sample analyses.

Number  of  patients     20  

Age  at  sampling  (median)     59.5  [26-­‐76]  

Sex   Male   9  

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*Survival  (median  months)     140  [61-­‐204]  

Status   Dead  with  disease   5  

  Alive  with  disease   15  

BMI  (median  kg/m2)     25.2  [20.4-­‐42.3]  

Systolic   blood   pressure   (median   mmHg)  

  145  [115-­‐180]  

Smoking  history  (n)   Current   3  

  Past   6     No   11   Antidepressants  (n)     3   Anti-­‐anxiaty  medication  (n)     3   Sleeping  pills  (n)     3   Diabetes  (n)     1  

Melatonin  (median  pg/L)     Sampling  1   24  [8-­‐115]  

  Sampling  2   23  [9-­‐609]  

U-­‐5HIAA  (median  μmol/24h)   Sampling  1   33  [5-­‐135]  

  Sampling  2   25  [5-­‐124]  

CgA  (median  nmol/L)   Sampling  1   3.25  [1.6-­‐66]  

  Sampling  2   3.8  [1.9-­‐43]  

*Survival is calculated as months from diagnosis to death

Values for BMI and systolic blood pressure are shown for the first sampling occasion.

U-5HIAA: Urinary 5-hydroxyindoleacetic acid (serotonin metabolite) normal reference value is 50 µmol/24h;

CgA: Chromogranin A, normal reference value is <4nmol/L;

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nor with survival or between plasma levels of melatonin and use of antidepressant/anti-anxiety drugs or the use of sleeping pills. No correlations were found between levels of melatonin and metabolic markers such as BMI, systolic blood pressure or plasma levels of glucose.

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Discussion  

Melatonin has been shown to exert a variety of peripheral functions, such as affecting gastrointestinal motility [68] and insulin secretion as well as protecting the gastrointestinal mucosa and inhibit proliferation in various types of cancer [58-61]. Unfortunately, the results for melatonin could not be completed in the time frame for this study due to methodological problems. However, this is the first study to our knowledge that demonstrates and characterizes the protein expression of both melatonin receptors (MT1 and MT2) in the different segments of human GIT. Our results are in agreement with findings of mRNA for both receptors in rat intestine [44], but contradictory to the findings for MT2 protein expression, where no expression was found in the mucosa [46]. Instead, protein expression was predominantly observed in the muscular layers of the GIT. Our results for MT1 confirm recent results where the MT1 receptor was identified in human colonic mucosa [27]. We further demonstrate MT2 expression in the majority of SI-NETs, which may provide a target for therapy.

In normal mucosa, positive expressions for MT1 as well as for MT2 were found in the epithelium, at all levels of intestinal segments studied.In the present study localization inside the epithelial cell varied and MT1 was mostly found in the cytoplasm, in the luminal peri-nuclear region, which is in agreement with the findings previously described [27]. Coinciding with localization of immune activity is the endoplasmatic reticulum and the Golgi apparatus, why it is conceivable to believe that signaling via the MT1 receptor is involved in production and packaging of proteins. The cytoplasmic localization of the MT1 receptor has previously been described in human mammary gland epithelial cells [33].

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been used to study behavior [76] and sleep-wake patterns [77]. Similar models could be used for studies of gastrointestinal function for these receptors.

For the MT2 receptor immunohistochemical activity was seen in cells deep in the crypts, which corresponds to endocrine cells, with localization of expression in the cytoplasm, in contrast to the localization for MT2 found in epithelial cells. Considering melatonin is known to have both paracrine, endocrine as well as autocrine properties, the activity found in endocrine cells could imply a feed-back mechanism for melatonin and its receptor. MT1 could not be found in endocrine cells to the same extent, except for one case where expression was only weakly positive.

SI-NETs are derived from enterochromaffin cells and these neurendocrine tumors generally have a relatively low proliferation rate. The expression of MT1 was rare and the signal was weak. A recent study of MT1 expression in colorectal adenocarcinoma, showed reduced expression of MT1 when compared to adjacent normal tissue which may indicate that MT1 could mediate the antitumorigenic effects [27]. Moreover, binding of melatonin to the MT1 receptor have been reported to up-regulate p27, a cyclin dependent kinase (Cdk) inhibitor protein, cell cycle regulator and tumor suppressor [62], that is expressed in areas of most SI-NETs [78].

In the tumor sections studied, MT2 was abundant. The intensity of the expression of MT2 was higher in primary tumors than in metastases and the difference was statistically significant. Expression levels in benign EC-cells in sections from normal gut appeared to be even stronger than in primary tumors which could imply that the more malignant the cell, the weaker the receptor expression. Melatonin is known to have antitumorigenic properties and inhibit proliferation in various types of cancer [58-61]. This loss of receptor expression could represent decreased sensitivity to the anti-proliferative properties of melatonin. MT2 levels did not however correlate to Ki67 Index, a proliferation marker. So the lower levels could be related to other properties of metastatic cells. Another alternative is that some factor in the mucosa stimulates MT2 expression and that the concentration decreases with the distance from the mucosa.

The antibody against MT2 was successfully neutralized (Fig. 1), which indicates specificity. For MT1, however, antibody neutralization was partial and the results for MT1 need to be verified. We have now obtained the correct peptide from the company and the neutralization experiments are underway.

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indicate that high levels are rare and not correlated with serotonin or CgA secretion from these tumors. Based on our hypothesis that SI-NETs would produce melatonin, higher levels ought to be expected. Oral administration of tryptophan, the precursor for melatonin, have been shown to increase plasma levels of melatonin [79], indicating that melatonin from the GIT represents a substantial part of circulating levels. One patient stood out with markedly higher values than the rest of the group, at both sampling occasions. This case was more thoroughly reviewed and described below.

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levels in females than in males. This trend consisted when using the Mann-Whitney test that compensates for outliers, but the difference was not statistically significant. As previously mentioned wide variety of factors may influence the secretion of gastrointestinal melatonin. It is related to periodicity of food intake and it can also be affected by high dietary content of its precursor tryptophan [11]. It is possible, perhaps even probable, that a lot of other confounding factors yet to be identified affect circulating levels of melatonin. The next step to further characterize this group of patients would be to increase the number of patients in the study and include a control group.

Considering the anti-inflammatory and oncostatic actions of melatonin that has so far been detected there is a great potential for melatonin to be used in future therapy. Many different medical conditions, including cardiovascular disease, gastrointestinal ulcus and cancer could be possible targets for such therapy why further extensive research is required in this field.

Conclusions  

Melatonin receptors, MT1 and MT2, were found to be present in human normal gastrointestinal mucosa from the stomach to the colon. The MT2 receptor was identified in endocrine cells in stomach, small intestine and large intestine. In SI-NETs the expression of MT2 was higher than the expression of MT1. For MT2 the expression was higher in primary tumor than in metastases.

Acknowledgements  

 

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References  

 

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