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Keywords:

  • DNA methyltransferase;
  • epigenetics;
  • melatonin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic and epigenetic regulation of genes
  5. DNMT inhibitors
  6. Basic facts about nucleoside DNMT inhibitors
  7. Non-nucleoside (small molecule) DNMT inhibitors
  8. A close look at the non-nucleoside (small molecule) DNMT inhibitors
  9. Cancers targeted in epigenetic therapy
  10. Acknowledgment
  11. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic and epigenetic regulation of genes
  5. DNMT inhibitors
  6. Basic facts about nucleoside DNMT inhibitors
  7. Non-nucleoside (small molecule) DNMT inhibitors
  8. A close look at the non-nucleoside (small molecule) DNMT inhibitors
  9. Cancers targeted in epigenetic therapy
  10. Acknowledgment
  11. References

Melatonin (N-acetyl-5-methoxytriptamine), the main secretory product of pineal gland is also produced by immune system cells and a number of peripheral tissues including brain, airway epithelium, bone marrow, gut, ovary, testes, skin, and others. Among functions which include anti-oxidative, anti-inflammatory, as well as the regulation of the daily and seasonal rhythms, melatonin may reduce the incidence and certainly the growth of tumors [1–4]. For melatonin to achieve these effects, it seems several mechanisms 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 of unstable oxygen and nitrogen-based reactants [5, 6]. Not only do oxygen and nitrogen-based reactants have the capability of disfiguring DNA which can lead to cancer initiation, but they are involved in tumor progression by activating signal transduction pathways and altering the expression of growth and differentiation-related genes [7]. Once tumors are formed, melatonin also seems to control their growth by other means, such as affecting the uptake and metabolism of fatty acids including linoleic acid [8], inhibiting telomerase activity [9], reducing endothelin-1 synthesis [10], and possibly others [2]. A recent randomized, controlled trial and meta-analysis confirmed the efficacy and safety of melatonin in cancer treatment [11]. Collectively, the findings to date uniformly suggest that melatonin is influential in inhibiting both cancer initiation and cancer cell growth.

Genetic and epigenetic regulation of genes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic and epigenetic regulation of genes
  5. DNMT inhibitors
  6. Basic facts about nucleoside DNMT inhibitors
  7. Non-nucleoside (small molecule) DNMT inhibitors
  8. A close look at the non-nucleoside (small molecule) DNMT inhibitors
  9. Cancers targeted in epigenetic therapy
  10. Acknowledgment
  11. References

As cancer is a growing problem in the modern world, novel treatments for such a debilitating disease have remained of major importance. Understanding the regulation of cellular proliferation and tumor development may help to uncover novel treatments for cancer. Tumor development is driven by selective forces that cause dysregulation of cellular proliferation. This is highlighted by the genetic and epigenetic inactivation of tumor suppressor genes in cancer cells. Not only genetic but also epigenetic mechanisms regulate 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 not only stable and heritable, but are also reversible [13]. They include covalent modifications of bases in the DNA and of amino acid residues in the histones. DNA methyltransferases (DNMTs) are a family of enzymes that methylate DNA at the carbon-5 position of cytosine residues. Methylated DNA can then be bound by methyl-binding proteins that function as adaptors between methylated DNA and chromatin-modifying enzymes (e.g. histone deacetylases and histone methyltransferases) by recruiting histone-modifying enzymes to patches of methylated DNA. Histone-modifying enzymes then covalently alter the amino-terminal residues of histones to induce the formation of chromatin structures that repress gene transcription [14]. Furthermore, DNMTs have been reported to be over-expressed in a variety of tumors. This might also contribute to the hypermethylation of tumor suppressor genes [15] (Fig. 1).

image

Figure 1.  Inhibitory mechanisms of non-nucleoside (small-molecule) DNA methyl transferase (DNMT) inhibitors (solid circles; methylated cytosine residues, open circles; demethylated or unmethylated cytosine residues). Physiologically, approximately 3–6% of the cytosine residues are methylated in mammals. Hypermethylation of cytosine residues in tumor suppressor genes by DNMTs cause gene silencing. Small-molecule inhibitors bind to the catalytic center of DNMTs and thereby inhibit DNA methylation directly. Demethylation can result in the reactivation of epigenetically silenced tumor suppressor genes. Drug removal or degradation leads to remethylation and resilencing. Small molecules can inhibit the enzyme by masking DNMT target sequences (i.e. procaine) or by blocking the active site of the enzyme (i.e. EGCG and RG108).

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It is now clear that genetic abnormalities found in cancers do not provide the complete picture of genomic alterations. Epigenetic changes, mainly DNA methylation and, more recently, modification of histones, are now recognized as additional mechanisms contributing to the malignant phenotype [16]. The study of these epigenetic changes on a genome-wide scale is referred to as epigenomics. Epigenetic modifications of DNA do not alter the sequence code; however, they are in heritable and are involved in regulation of gene transcription. DNA methylation, the addition of a methyl group to cytosine, is one such epigenetic modification found in DNA. DNA methylation is a dynamic but tightly regulated process. Methylation patterns are faithfully transmitted to the next generation during cell division, yet, during embryonic development, currently undefined regulatory mechanisms allow rapid demethylation in very early stages followed by re-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 the components of this regulatory network, let alone the organization and role of each of the components.

Genomic tumor DNA is generally characterized by distinct methylation changes that have also been termed epimutations [16]. At the global level, DNA is often hypomethylated, particularly at centromeric repeat sequences; this hypomethylation has been linked to genomic instability. Another class of epimutations is characterized by the local hypermethylation of individual genes; this is associated with aberrant gene silencing.

Currently, it is believed that hypermethylation and epigenetic silencing of tumor suppressor genes play important roles in the etiology of human cancers. In contrast to DNA mutations, which are passively inherited through DNA replication, epimutations must be actively maintained because they are reversible [17]. Such epimutations rarely appear in healthy tissue, indicating that epigenetic therapies may have high tumor specificity.

The reversibility of epigenetic modifications renders them attractive targets for therapeutic interventions. In contrast to genetic mutations, which are inherited passively through DNA replication, epigenetic mutations must be actively maintained. Consequently, pharmacologic inhibition of certain epigenetic modifications could correct faulty modification patterns and thus, directly change gene expression patterns and the corresponding cellular characteristics. As hypermethylation and epigenetic silencing of tumor suppressor genes have gained importance in the etiology of human cancer, the pharmacological inhibition of DNMTs provides a novel opportunity for the therapy of human cancers [14].

DNMT inhibitors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic and epigenetic regulation of genes
  5. DNMT inhibitors
  6. Basic facts about nucleoside DNMT inhibitors
  7. Non-nucleoside (small molecule) DNMT inhibitors
  8. A close look at the non-nucleoside (small molecule) DNMT inhibitors
  9. Cancers targeted in epigenetic therapy
  10. Acknowledgment
  11. References

Progress in the development of pharmacologic DNMTs inhibitors has been confirmed in phase I–III clinical trials. In addition, the prototypical DNMT inhibitor 5-azacytidine (i.e. Vidaza) has recently been approved by the US Food and Drug Administration as an antitumor agent for the treatment of myelodysplastic syndrome. There are two types of DNMTs inhibitors, namely, nucleoside and non-nucleoside (small molecule) inhibitors [12].

Basic facts about nucleoside DNMT inhibitors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic and epigenetic regulation of genes
  5. DNMT inhibitors
  6. Basic facts about nucleoside DNMT inhibitors
  7. Non-nucleoside (small molecule) DNMT inhibitors
  8. A close look at the non-nucleoside (small molecule) DNMT inhibitors
  9. Cancers targeted in epigenetic therapy
  10. Acknowledgment
  11. References

The archetypal nucleoside DNMT inhibitor is 5-azacytidine, a simple derivative of the nucleoside cytidine [18]. Its demethylating activity was discovered as the result of its ability to influence cellular differentiation. 5-Azacytidine is a nucleoside inhibitor that is incorporated into DNA. DNMTs methylate both cytosine residues and 5-azacytosine residues in DNA. However, 5-azacytosine prevents the resolution of a covalent reaction intermediate, which leads to the DNMT being trapped and inactivated in the form of a covalent protein–DNA adduct [14]. As a result, cellular DNMTs are rapidly depleted, and concomitantly genomic DNA is demethylated as a result of continued DNA replication.

Non-nucleoside (small molecule) DNMT inhibitors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic and epigenetic regulation of genes
  5. DNMT inhibitors
  6. Basic facts about nucleoside DNMT inhibitors
  7. Non-nucleoside (small molecule) DNMT inhibitors
  8. A close look at the non-nucleoside (small molecule) DNMT inhibitors
  9. Cancers targeted in epigenetic therapy
  10. Acknowledgment
  11. References

Some non-nucleoside compounds can also inhibit DNMT activity. 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-gallate (EGCG) [19], a major polyphenol compound in green tea. EGCG affects various biologic pathways and inhibits DNMT activity in protein extracts and in human cancer cell lines. Another pharmacologically developed DNMT 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 but that have few or no side effects and a wide safety margin. A major advantage of this approach is a well-known pharmacodynamic profile of the respective drugs and their cost-efficient adaptation to oncologic use. Examples of these compounds include the antihypertensive drug hydralazine, the local anesthetic procaine, and the antiarrhythmic drug procainamide [21]. A second strategy is the rational design of small molecules that block the active site of human DNMTs such as RG108. This approach is more cost intensive, but it could result in the development of highly specific drugs.

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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic and epigenetic regulation of genes
  5. DNMT inhibitors
  6. Basic facts about nucleoside DNMT inhibitors
  7. Non-nucleoside (small molecule) DNMT inhibitors
  8. A close look at the non-nucleoside (small molecule) DNMT inhibitors
  9. Cancers targeted in epigenetic therapy
  10. Acknowledgment
  11. References

The non-nucleoside DNMT inhibitors have been proposed to 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 an interesting structural similarity.

Melatonin and its metabolites [22] have a similar structure and hypothetically could inhibit DNMT either by masking target sequences or by blocking the active site of the enzyme. Melatonin is a highly lipophilic and somewhat hydrophilic molecule that easily crosses cell membranes reaching intracellular organelles including the nucleus [23]. Melatonin may accumulate in the nucleus and it interacts with specific nuclear binding sites [24]. So-called nuclear receptors for melatonin have been identified and some studies have linked them to melatonin’s control of cell growth and differentiation [25]. Melatonin has a long-shelf life and has few or no side effects [26]. Not only melatonin itself, but also many of its derivatives and metabolites are biologically active [22, 27]. Several derivatives of melatonin are produced in the intracellular environment when the indoleamine scavenges reactive oxygen and nitrogen species. Because of higher metabolic rates, cancer cells typically generate increased numbers of reactive oxygen and nitrogen species [28]. Melatonin first scavenges these toxic compounds and is then converted to active metabolites including cyclic 3-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine, and others (Fig. 1) [22, 29]. Melatonin may be readily converted to these metabolites in cancer cells which, along with melatonin itself, may exert DNMT inhibitory effects.

Cancers targeted in epigenetic therapy

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic and epigenetic regulation of genes
  5. DNMT inhibitors
  6. Basic facts about nucleoside DNMT inhibitors
  7. Non-nucleoside (small molecule) DNMT inhibitors
  8. A close look at the non-nucleoside (small molecule) DNMT inhibitors
  9. Cancers targeted in epigenetic therapy
  10. Acknowledgment
  11. References

Abnormal DNA methylation patterns or epimutations have been documented for various cancers. These epimutations may be used as biomarkers for tumor classification. Epimutations appear to accumulate over time at various sites in the genome and to promote tumorigenesis by increasing genomic instability or by silencing tumor suppressor genes. The silencing of tumor suppressor genes is closely associated with DNA hypermethylation and can be effectively reversed by DNMT inhibitors. For these reasons, non-nucleoside DNMT inhibitors may be an attractive 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 cell lines. Tumors frequently show an increase in matrix metalloproteinase (MMP) and/or a decrease in tissue inhibitor metalloproteinase-3 (TIMP-3) leading to an imbalance in proteolytic activity during tumor progression. TIMP-3 is a secreted 24-kDa protein which, binds to the extracellular matrix. TIMP-3 antagonizes the activity of MMPs by binding covalently to the active site of the enzymes. It is thought that reduced expression of TIMP-3 contributes to primary tumor growth, angiogenesis, apoptosis, tumor invasion, and metastasis by allowing increased activity of MMPs in the extracellular matrix. Recent studies on methylation-associated silencing of TIMP-3 suggest a tumor suppressor role in kidney, brain, breast, and colon cancers [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 investigated at the genomic DNA level using methylation-specific PCR or capillary electrophoretic analysis, mRNA levels with the aid of RT-PCR and/or examining protein levels using immunohistochemical staining.

Given that epigenetic modifications are also responsible for several diseases in addition to cancer, melatonin’s epigenetic efficacy may appear in hypertension [31] and in inheritance of environmental changes during pregnancy [32]. Inhibition of telomerase [9], endothelin-1 [10] and in a recent study, TIMPs/MMPs activity during prevention of ethanol-induced gastric ulcer [33] in mice, may also involve epigenetic regulation. Based on currently available information, a novel research area for mechanisms of cancer inhibition by melatonin may have emerged. Readers may be encouraged to investigate this novel research area.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Genetic and epigenetic regulation of genes
  5. DNMT inhibitors
  6. Basic facts about nucleoside DNMT inhibitors
  7. Non-nucleoside (small molecule) DNMT inhibitors
  8. A close look at the non-nucleoside (small molecule) DNMT inhibitors
  9. Cancers targeted in epigenetic therapy
  10. Acknowledgment
  11. References