MINI REVIEW

Epigenetic regulation: a new research area for melatonin?

Introduction

Melatonin (N-acetyl-5-methoxytriptamine), the main secre-tory product of pineal gland is also produced by immune

system cells and a number of peripheral tissues includingbrain, airway epithelium, bone marrow, gut, ovary, testes,skin, and others. Among functions which include anti-

oxidative, anti-inflammatory, as well as the regulation ofthe daily and seasonal rhythms, melatonin may reduce theincidence and certainly the growth of tumors [1–4]. For

melatonin to achieve these effects, it seems several mech-anisms are involved.

In terms of limiting the frequency of cancer initiation,one of the mechanisms may be the ability of melatonin to

reduce severe DNA damage that is a consequence ofunstable oxygen and nitrogen-based reactants [5, 6]. Notonly do oxygen and nitrogen-based reactants have the

capability of disfiguring DNA which can lead to cancerinitiation, but they are involved in tumor progression byactivating signal transduction pathways and altering the

expression of growth and differentiation-related genes [7].Once tumors are formed, melatonin also seems to controltheir growth by other means, such as affecting the uptakeand metabolism of fatty acids including linoleic acid [8],

inhibiting telomerase activity [9], reducing endothelin-1synthesis [10], and possibly others [2]. A recent randomized,controlled trial and meta-analysis confirmed the efficacy

and safety of melatonin in cancer treatment [11]. Collec-tively, the findings to date uniformly suggest that melatoninis influential in inhibiting both cancer initiation and cancer

cell growth.

Genetic and epigenetic regulation of genes

As cancer is a growing problem in the modern world, noveltreatments for such a debilitating disease have remained of

major importance. Understanding the regulation of cellularproliferation and tumor development may help to uncovernovel treatments for cancer. Tumor development is driven

by selective forces that cause dysregulation of cellularproliferation. This is highlighted by the genetic andepigenetic inactivation of tumor suppressor genes in cancer

cells. Not only genetic but also epigenetic mechanismsregulate the expression of genetic information.Epigenetics is the study of heritable changes in gene

expression that are not encoded in the DNA sequence itself

[12]. Epigenetic modifications of DNA and histones are notonly stable and heritable, but are also reversible [13]. Theyinclude covalent modifications of bases in the DNA and of

amino acid residues in the histones. DNA methyltransfe-rases (DNMTs) are a family of enzymes that methylateDNA at the carbon-5 position of cytosine residues. Methy-

lated DNA can then be bound by methyl-binding proteinsthat function as adaptors between methylated DNA andchromatin-modifying enzymes (e.g. histone deacetylasesand histone methyltransferases) by recruiting histone-mod-

ifying enzymes to patches of methylated DNA. Histone-modifying enzymes then covalently alter the amino-terminalresidues of histones to induce the formation of chromatin

structures that repress gene transcription [14]. Furthermore,DNMTs have been reported to be over-expressed in avariety of tumors. This might also contribute to the

hypermethylation of tumor suppressor genes [15] (Fig. 1).

Abstract: Epigenetic, modifications of DNA and histones, i.e. heritable

alterations in gene expression that do not involve changes in DNA sequences,

are known to be involved in disease. Two important epigenetic changes that

contribute to disease are abnormal methylation patterns of DNA and

modifications of histones in chromatin. Epimutations, such as the

hypermethylation and epigenetic silencing of tumor suppressor genes, have

revealed a new area for cancer treatment. Studies using DNA

methyltransferase inhibitors such as procaine, hydralazine, and RG108 have

had promising outcomes against cancer therapy. Melatonin, one of the most

versatile molecules in nature, may hypothetically be involved in epigenetic

regulation. In this review, the potential role of melatonin in inhibiting DNA

methyltransferase and epigenetic regulation is discussed.

Ahmet Korkmaz1 and Russel J.Reiter 2

1Department of Physiology, School of

Medicine, Gulhane Military Medical Academy,

Ankara, Turkey; 2Department of Cellular and

Structural Biology, The University of Texas

Health Science Center at San Antonio, San

Antonio, TX, USA

Key words: DNA methyltransferase,

epigenetics, melatonin

Address reprint requests to Ahmet Korkmaz,

Department of Physiology, School of Medicine,

Gulhane Military Medical Academy, 06018

Etlik, Ankara, Turkey.

E-mail: fizyocan@gmail.com

Received August 20, 2007;

accepted August 28, 2007.

J. Pineal Res. 2008; 44:41–44Doi:10.1111/j.1600-079X.2007.00509.x

� 2007 The AuthorsJournal compilation � 2007 Blackwell Munksgaard

Journal of Pineal Research

41

It is now clear that genetic abnormalities found incancers do not provide the complete picture of genomic

alterations. Epigenetic changes, mainly DNA methylationand, more recently, modification of histones, are nowrecognized as additional mechanisms contributing to the

malignant phenotype [16]. The study of these epigeneticchanges on a genome-wide scale is referred to as epige-nomics. Epigenetic modifications of DNA do not alter thesequence code; however, they are in heritable and are

involved in regulation of gene transcription. DNA methyl-ation, the addition of a methyl group to cytosine, is onesuch epigenetic modification found in DNA. DNA meth-

ylation is a dynamic but tightly regulated process. Meth-ylation patterns are faithfully transmitted to the nextgeneration during cell division, yet, during embryonic

development, currently undefined regulatory mechanismsallow rapid demethylation in very early stages followed byre-establishment of methylation patterns after implantation[17]. While some of the enzymes involved in these processes

are known, there is only a basic understanding of thecomponents of this regulatory network, let alone theorganization and role of each of the components.

Genomic tumor DNA is generally characterized bydistinct methylation changes that have also been termedepimutations [16]. At the global level, DNA is often

hypomethylated, particularly at centromeric repeatsequences; this hypomethylation has been linked to geno-mic instability. Another class of epimutations is character-

ized by the local hypermethylation of individual genes; thisis associated with aberrant gene silencing.Currently, it is believed that hypermethylation and

epigenetic silencing of tumor suppressor genes play impor-

tant roles in the etiology of human cancers. In contrast toDNA mutations, which are passively inherited throughDNA replication, epimutations must be actively maintained

because they are reversible [17]. Such epimutations rarelyappear in healthy tissue, indicating that epigenetic therapiesmay have high tumor specificity.

The reversibility of epigenetic modifications renders themattractive targets for therapeutic interventions. In contrast

to genetic mutations, which are inherited passively throughDNA replication, epigenetic mutations must be activelymaintained. Consequently, pharmacologic inhibition of

certain epigenetic modifications could correct faulty mod-ification patterns and thus, directly change gene expressionpatterns and the corresponding cellular characteristics. Ashypermethylation and epigenetic silencing of tumor sup-

pressor genes have gained importance in the etiology ofhuman cancer, the pharmacological inhibition of DNMTsprovides a novel opportunity for the therapy of human

cancers [14].

DNMT inhibitors

Progress in the development of pharmacologic DNMTsinhibitors has been confirmed in phase I–III clinical trials.In addition, the prototypical DNMT inhibitor 5-azacyti-

dine (i.e. Vidaza) has recently been approved by the USFood and Drug Administration as an antitumor agent forthe treatment of myelodysplastic syndrome. There are two

types of DNMTs inhibitors, namely, nucleoside and non-nucleoside (small molecule) inhibitors [12].

Basic facts about nucleoside DNMTinhibitors

The archetypal nucleoside DNMT inhibitor is 5-azacyti-dine, a simple derivative of the nucleoside cytidine [18]. Itsdemethylating activity was discovered as the result of itsability to influence cellular differentiation. 5-Azacytidine is

a nucleoside inhibitor that is incorporated into DNA.DNMTs methylate both cytosine residues and 5-azacyto-sine residues in DNA. However, 5-azacytosine prevents the

resolution of a covalent reaction intermediate, which leadsto the DNMT being trapped and inactivated in the form ofa covalent protein–DNA adduct [14]. As a result, cellular

Fig. 1. Inhibitory mechanisms of non-nucleoside (small-molecule) DNA methyltransferase (DNMT) inhibitors (solid cir-cles; methylated cytosine residues, opencircles; demethylated or unmethylatedcytosine residues). Physiologically, appro-ximately 3–6% of the cytosine residues aremethylated in mammals. Hypermethy-lation of cytosine residues in tumor sup-pressor genes by DNMTs cause genesilencing. Small-molecule inhibitors bindto the catalytic center of DNMTs andthereby inhibit DNA methylation directly.Demethylation can result in the reactiva-tion of epigenetically silenced tumorsuppressor genes. Drug removal ordegradation leads to remethylation andresilencing. Small molecules can inhibitthe enzyme by masking DNMT targetsequences (i.e. procaine) or by blockingthe active site of the enzyme (i.e. EGCGand RG108).

Korkmaz and Reiter

42

DNMTs are rapidly depleted, and concomitantly genomicDNA is demethylated as a result of continued DNAreplication.

Non-nucleoside (small molecule) DNMTinhibitors

Some non-nucleoside compounds can also inhibit DNMTactivity. These substances directly block DNMTs and,therefore, do not appear to have the inherent toxicity

caused by the covalent trapping of the enzyme. One non-nucleoside DNMT inhibitor is ())-epigallocatechin-3-gal-late (EGCG) [19], a major polyphenol compound in green

tea. EGCG affects various biologic pathways and inhibitsDNMT activity in protein extracts and in humancancer cell lines. Another pharmacologically developedDNMT inhibitor, so-called RG108, blocks the active site of

DNMT [20].To uncover alternative DNMT inhibitors, two strategies

are being used. The first strategy is the exploitation of

established chemicals that have already been approved butthat have few or no side effects and a wide safety margin. Amajor advantage of this approach is a well-known phar-

macodynamic profile of the respective drugs and their cost-efficient adaptation to oncologic use. Examples of thesecompounds include the antihypertensive drug hydralazine,

the local anesthetic procaine, and the antiarrhythmic drugprocainamide [21]. A second strategy is the rational designof small molecules that block the active site of humanDNMTs such as RG108. This approach is more cost

intensive, but it could result in the development of highlyspecific drugs.

A close look at the non-nucleoside (smallmolecule) DNMT inhibitors

The non-nucleoside DNMT inhibitors have been proposedto suppress DNMTs by masking DNMT target sequences(i.e. procaine) or by blocking the active site of the enzyme(i.e. EGCG and RG108). A closer look at the non-

nucleoside small molecule DNMT inhibitors reveals aninteresting structural similarity.

Melatonin and its metabolites [22] have a similar

structure and hypothetically could inhibit DNMT eitherby masking target sequences or by blocking the active siteof the enzyme. Melatonin is a highly lipophilic and

somewhat hydrophilic molecule that easily crosses cellmembranes reaching intracellular organelles including thenucleus [23]. Melatonin may accumulate in the nucleus and

it interacts with specific nuclear binding sites [24]. So-callednuclear receptors for melatonin have been identified andsome studies have linked them to melatonin�s control of cellgrowth and differentiation [25]. Melatonin has a long-shelf

life and has few or no side effects [26]. Not only melatoninitself, but also many of its derivatives and metabolites arebiologically active [22, 27]. Several derivatives of melatonin

are produced in the intracellular environment when theindoleamine scavenges reactive oxygen and nitrogen spe-cies. Because of higher metabolic rates, cancer cells

typically generate increased numbers of reactive oxygenand nitrogen species [28]. Melatonin first scavenges

these toxic compounds and is then converted to activemetabolites including cyclic 3-hydroxymelatonin, N1-ace-tyl-N2-formyl-5-methoxykynuramine, and others (Fig. 1)

[22, 29]. Melatonin may be readily converted to thesemetabolites in cancer cells which, along with melatoninitself, may exert DNMT inhibitory effects.

Cancers targeted in epigenetic therapy

Abnormal DNA methylation patterns or epimutations have

been documented for various cancers. These epimutationsmay be used as biomarkers for tumor classification.Epimutations appear to accumulate over time at various

sites in the genome and to promote tumorigenesis byincreasing genomic instability or by silencing tumorsuppressor genes. The silencing of tumor suppressor genesis closely associated with DNA hypermethylation and can

be effectively reversed by DNMT inhibitors. For thesereasons, non-nucleoside DNMT inhibitors may be anattractive treatment option for most tumors either alone

or in combination with other chemotherapeutic drugs.The initial test to reveal if melatonin has DNMT

inhibitory and/or other epigenetic effects could be examined

both in cell-free in vitro systems and/or human cancer celllines. Tumors frequently show an increase in matrixmetalloproteinase (MMP) and/or a decrease in tissue

inhibitor metalloproteinase-3 (TIMP-3) leading to animbalance in proteolytic activity during tumor progression.TIMP-3 is a secreted 24-kDa protein which, binds to theextracellular matrix. TIMP-3 antagonizes the activity of

MMPs by binding covalently to the active site of theenzymes. It is thought that reduced expression of TIMP-3contributes to primary tumor growth, angiogenesis, apop-

tosis, tumor invasion, and metastasis by allowing increasedactivity of MMPs in the extracellular matrix. Recent studieson methylation-associated silencing of TIMP-3 suggest a

tumor suppressor role in kidney, brain, breast, and coloncancers [30].Changes in TIMP-3 and MMPs levels in cell cultures as a

result of melatonin treatment would support the epigenetic

efficacy of this molecule. These proteins could be investi-gated at the genomic DNA level using methylation-specificPCR or capillary electrophoretic analysis, mRNA levels

with the aid of RT-PCR and/or examining protein levelsusing immunohistochemical staining.Given that epigenetic modifications are also responsible

for several diseases in addition to cancer, melatonin�sepigenetic efficacy may appear in hypertension [31] and ininheritance of environmental changes during pregnancy

[32]. Inhibition of telomerase [9], endothelin-1 [10] and in arecent study, TIMPs/MMPs activity during prevention ofethanol-induced gastric ulcer [33] in mice, may also involveepigenetic regulation. Based on currently available infor-

mation, a novel research area for mechanisms of cancerinhibition by melatonin may have emerged. Readers may beencouraged to investigate this novel research area.

Acknowledgment

The authors would like to thank Dr Turgut Topal fordrawing the demonstrative figure.

Melatonin and epigenetic regulation

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