Department of Neuroscience and Psychiatry

Supervisor: Janet Cunningham

 

 

 

 

Melatonin  receptor  expression  in  the  

normal  human  gastrointestinal  tract  and  

in  gastrointestinal  neuroendocrine  

tumors  

 

Fanny  Söderquist  

  2  

 

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).

Receptor expression indicates a role for melatonin signaling in the epithelium and in

endocrine cells of the GIT. Melatonin has well documented anti-proliferative effects

and melatonin receptor expression in tumors may provide a target for therapy.

  3  

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).

Uttrycket av bade MT1 och MT2 i mag- tarmkanalen indikerar att melatonin är

involverat i signalering i epitelet och i endokrina celler. Melatonin har

väldokumenterade antiproliferativa effekter och uttrycket av melatoninreceptorer i

tumörer kan utgöra ett mål för behandling.

  4  

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]  

Immune  system   MT1,  MT2  (malignant   PCR,  mRNA   [21]  [22]  

  5  

(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

results indicate that the MT2 receptor subtype might be present also in human

duodenum, as the effect was abolished by an MT2-selective receptor antagonist [43].

In rats, mRNA transcripts of both MT1 and MT2 have been detected in the small

intestine [44] and MT1 mRNA was expressed in the small intestine and colon with

the highest expression in the sub epithelial layers (muscularis externa and serosa) of

the duodenum [45]. The distribution of the MT2 receptor was characterized using

immunohistochemistry and western blot. The highest density was found in the colon,

  6  

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].

The effects of melatonin in the GIT include decreased intestinal motility by relaxing

smooth muscle [68]. It appears to act as a physiological antagonist of serotonin in the

  7  

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.

The aim of the study is to map the tissue expression of melatonin and its receptors

in normal gut and in SI-NETs. A secondary aim is to look for correlations between

levels of circulating melatonin and use of psychiatric drugs and markers of metabolic

status in patients with SI-NETs.

  8  

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).

The study was approved by the local ethics committee of the Uppsala University

  9  

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.

The immunostained sections from normal tissue represented stomach (n=5), small

  10  

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.

For statistical analyses of differences between the grading of primary tumors and

metastases, the scoring (0-3) were regrouped into two outcomes; low expression

which was the negative and the weaker staining of grade 1 and high expression which

was the stronger staining of grade 2-3. Fisher’s exact test was used to evaluate the

difference. Results are presented as median [range] unless otherwise is stated.

  11  

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  

Positive immunohistochemical staining of MT1 was found primarily in epithelial cells

lining the crypts. In the large intestine the MT1 receptor was found in all sections

studied. Localization was cytoplasmic, mainly peri-nuclear towards the lumen.

Intensity varied with different sections and there were differences in the level of the

  12  

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.

 

 

  13  

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  

  Past   10  

  14  

  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.

No significant association was found between MT1 or MT2 receptor staining and

BMI, systolic blood pressure, use of psychiatric drugs or survival.  

  15  

 

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

 

 

 

  16  

 

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  

  Female   11  

  17  

*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;

Median plasma level of melatonin at the first sampling, before interferon treatment,

was 24 pg/L [8-115] and 3 months after initiation of interferon treatment, median

plasma level was pg/L [9-609] (Fig. 6).  The same individual represented the highest

values at both sampling occasions. Levels were higher in females than in males, but

the difference was not statistically significant (p=0.271). Interferon treatment did not

influence melatonin levels. There was no significant correlation between plasma

levels of melatonin and other markers for hormone production U-5HIAA and CgA,

  18  

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.

Fig. 6. Plasma levels of melatonin measured at two sampling occasions. Values are

displayed on a logarithmic scale.

  19  

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].

The expression of the MT2 receptor in epithelial cells was most prominent in the

nuclei, which would seem unexpected since the receptor is a GPCR that ought to be

membrane-bound. Nuclear expression of the MT2 receptor subtype has previously

been described in human placental tissues [73] and recently other known GPCRs have

been reported to have nuclear localization [74]. Melatonin is lipophilic and can easily

diffuse through cell membranes to access an intracellular receptor. This could indicate

another way of signaling or involvement in gene transcription, since it has been

shown that melatonin, in a receptor-dependent manner can influence transcription

factors involved in insulin secretion [75]. Melatonin receptor knock-out mice have

  20  

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.

Plasma levels of melatonin were analyzed in patients with SI-NETs and the results

  21  

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.

The patient with the highest levels of melatonin was a 45-y-old, postmenopausal,

previously smoking female patient with hypertension, chronic IgA-glomerulonephritis

and modest chronic autoimmune hepatitis. In addition to antihypertensive medication

the patient used sleeping pills (Stilnoct) intermittently. The patient’s body mass

index (BMI) was 33.8 kg/m2 and there was a 3-year history of loose stools,

postprandial abdominal pains and facial flushing after drinking wine reported in

clinical records. During the year prior to diagnosis, the patient had sought medical

care repeatedly because of abdominal pain and gastrointestinal bleeding; no source of

bleeding was found. The diagnosis of midgut carcinoid was made when the patient

fell ill with ileus and underwent acute laparotomy. A primary tumor of 2.5 cm was

found, infiltrating the muscularis propria, as well as metastases to the lymph nodes.

Ki67 was <0.5%. A second operation was performed with the removal of lymph node

metastases. Interferon treatment was started but was canceled due to elevated liver

enzymes. No apparent differences in the patient’s history versus the rest of the group

were found that could explain the greatly elevated levels of melatonin in plasma. The

case is interesting since it raises the question of why levels of melatonin were so

elevated in this patient. If it was the tumor that produces the melatonin that represent

the elevation, then why was there a difference in the group? Perhaps there are some

subpopulations of SI-NETs that do produce melatonin while others do not. It was

noted in the present study that endocrine cells positive for serotonin were more

numerous than those positive for the MT2 receptor in matched sections, hence,

subpopulations of endocrine cells and endocrine tumors may exist. Another

possibility is that the high melatonin levels are in some way related to the

autoimmune disorders. This is not previously described in the literature but melatonin

does regulate autoimmunity [80].

Another observation in the plasma levels of melatonin was a trend towards higher

  22  

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  

 

I would particularly like to thank my supervisor Janet Cunningham, MD for all the

support and inspiration, Åsa Forsberg, laboratory technician for excellent technical

assistance, and Hans Arinell, statician at the Departement of Neurosience and

Psychiatry for his assistance in the statistical analysis of the data.

  23  

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