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DNA methylation

  1. 1. Send Orders for Reprints to Epigenetic Diagnosis & Therapy, 2015, 1, 5-13 5 2214-0840/15 $58.00+.00 © 2015 Bentham Science Publishers DNA Methylation: A Possible Target for Current and Future Studies on Cancer? Sahar Olsadat Sajadian and Andreas K. Nussler* University of Tübingen, BG Trauma Center, Siegfried Weller Institute, Schnarrenbergstraße 95, 72076 Tübingen, Germany Abstract: The field of epigenetics encompasses studies about the ways in which genes are influenced by various external stimuli, such as drugs, chemicals, and the environment. Over the last few years, it has become an increasingly important area of research. Recently, substantial evidence has been presented for the claim that certain diseases, including cancer, exhibit epigenetic changes. In the present overview, we will give a brief summary of recent observations on epigenetics and cancer as well as of the ways in which various small molecules may influence the epigenetic state of cells. Keywords: Cancer, CpG islands, DNA methylation, epigenetic drugs, epigenetics, TET proteins. DEFINITION OF EPIGENETICS Epigenetics is defined ‘as a permanent change of gene expression during cell proliferation and development, which does not cause a change of the gene sequences’. Epigenetic regulation takes place in three separate stages: 1) nucleosome positioning, 2) histone modification, and 3) DNA methylation (Fig 1). DNA METHYLATION In the past years, epigenetic studies have focused on DNA methylation in mammals. Particular interest was directed towards genomic imprinting in the inactivation pattern of X-chromosome, in silencing of the retrotransposon, known as repetitive elements, and in tissue- specific genes [1, 2], These epigenetic changes have demonstrated playing an important role in many pathologies and gene therapies [3-5]. During the DNA methylation process, a methyl group (CH3) is added to the phenyl ring of the cytosine at the 5- carbon position. Somatic cell methylation can take place at all CpG (cytosine- phosphate-guanin) dinucleotides positions except at the CpG island [6]. CpG islands are parts of the DNA with more than 500 bp and over 55% of GC content [7]. These islands accommodate approximately 60% of the mammalian gene promoters [8]. When DNA methylation occurs in these promoters, some crucial transcription factors (TF) are down-regulated with subsequent inhibition of the corresponding protein formation. However, it has been proven that even in regions that are poor in CpGs, such as the Oct4 promoter, DNA methylation plays an important role in gene regulation [9, 10]. *Address correspondence to this author at the University of Tübingen, BG Trauma Center, Siegfried Weller Institute, Schnarrenbergstraße 95, D-72076 Tübingen, Germany, Tel: +49 7071 606-1065; Fax: +49 7071 606-1978; E-mail: DNA methylation is supported by a special group of enzymes; the so called DNA methyltransferases (DNMT). Up to now, the following DNMTs have been discovered: DNMT1, DNMT1b, DNMT1o, DNMT1p, DNMT2, DNMT3A, DNMT3B, and DNMT3L. DNMTs such as DNMT3A, DNMT3B, and DNMT3L are apparently responsible for the de novo DNA methylation in early developmental stages. In order to maintain the methylation in daughter cells, the so called UHRF1 protein, a ubiquitin-like protein with PHD and RING finger domain 1, binds to hemi-methylated DNA. During replications, UHRF1 causes DNMT1 to methylate new cytosine from synthesized DNA strands. There exists also growing evidence which suggests that DNMT3A/B are also involved in maintaining the DNA methylation pattern in somatic cells, specifically in imprinted genes and repeated elements [10]. The significance of DNMTs was demonstrated in various animal studies, proving that mice lacking DNMTs died during their early development or shortly after birth [11]. Nevertheless, the expression of these enzymes differs between certain types of cancer and it is not always associated with hyper- or hypomethylation, as had been expected. Sometimes, the expression of DNMTs is regulated by miRNAs, which may explain the differences in the expression of DNMTs in various tumors [12]. For instance, it has been demonstrated in several studies that DNA demethylase is post-transcriptionally regulated by mir29 and mir148 [13]. Furthermore, So et al. have claimed that DNA methyltransferase controls stem cell aging through microRNA. They have shown that DNMT inhibition leads to a decrease of the expression of Polycomb groups (PcG) and to an increase of the expression of microRNAs (miRNAs) that target PcG [14]. DNA DEMETHYLATION In early embryogenesis, many researchers have observed a loss of DNA methylation and subsequently have postulated the existence of several DNA demethylases and various
  2. 2. 6 Epigenetic Diagnosis & Therapy, 2015, Vol. 1, No. 1 Sajadian and Nussler mechanisms of the DNA demethylation [15, 16]. One potential DNA demethylation pathway is enabled by the ten- eleven-translocation proteins (TETs), which are named after the ten-eleven-translocation (q22, q23) (10, 11) that occurs in rare cases of acute myeloid and lymphocytic leukemia [17, 18]. 5-mC is oxidized by TET proteins and thereby converted to 5-hmC, 5-fC, and 5-CaC, which all belong to the family of Fe (II)/2-oxygluttarate-dependent dioxgenases [19]. DNA methylation is reversible either through passive demethylation via DNA replication or by active demethylation via DNA repair followed by base excision repair of modified nucleotides or DNA glycosidase [15, 17] (Fig. 2). TET proteins play different roles in diverse biological processes, including epigenetic regulation of gene transcription, embryonic development, stem cell functions, and cancer. They mediate the DNA demethylation process and change the epigenetic state of DNA. A loss of 5-hmC, together with a down-regulation or mutation of TET proteins, has been reported in numerous types of cancer, like melanoma, breast, prostate, myeloid, brain, and liver cancer [20]. It has been demonstrated that in patients suffering from acute myeloid leukemia (AML), TET1 protein influences mixed lineage leukemia (MLL) development [16]. Furthermore, several changes in TET2 proteins, like e.g. deletion and deactivation of TET2, have been identified in 30% of all patients suffering from different kinds of myeloid malignancies. Several work groups have reported an alteration of a TET2 gene in a variety of myeloid malignancies and the majority of the TET2 alterations include single or double copy defects and nonsense/frame shift mutation suggesting that TET2 lesions cause a loss of function. Therefore, it has been suggested that TET2 is a possible tumor suppressor gene that is responsible for myeloid malignancies [21]. Furthermore, the loss of 5-hmC in correlation with clinical progression and relapse of melanoma has been reported [20]. DNA METHYLATION AND CANCER Numerous studies have reported that tumor cells exhibit a major disruption in the regulation of the DNA methylation pattern in cancer. Therefore, over the last few years, studies have particularly focused on changes in DNA methylation. [22]. Earlier studies have demonstrated that the hypomethylation of interspersed repetitive DNA is a common feature of numerous types of cancer that causes genomic instability and aberrant transcription initiation. However, researchers have proven that the hypermethylation of focal CpG islands is elevated in cancer and may result in Fig. (1). Epigenetic machinery and interplay between epigenetic factors (modified from [91]). Epigenetic regulation depends on the three following epigenetic factors: (a) DNA methylation, (b) histone modification, and (c) nucleosome positioning. The interaction between these factors results in the final outcome, but disturbances of this balance lead to several diseases. Abbreviations: DNMT: DNA Methyl Transferase, HAT: Histone Acetylation Transferase, HDAC: Histone Deacetylase, HMT: Histone Methyltransferase, HDM: Histone Demethyl Transferase. MethylationMethylation DemethylationDemethylation DNMT1 TETs: Me DNA Methylation (maintenance DNMT) DNMT3A & DNMT3B (de novo DNMT) TET1, TET2, TET3 5hmC Epigenetic machinery Epigenetic machinery AcetylationAcetylation HAT & HDAC Histone modifications Histone modifications Chromatin remodeling Chromatin remodeling MethylationMethylation HAT & HDAC HMT & HDM Disfunc Phosphor- tylation Phosphor- tylation Kinase & Phosphor SWI/SNF, ISWI MI-2, INO80 Disease ctioning Ac Me Me P Disease • Cancer • Autoimmune disorder • Neural disorder • Aging Ub
  3. 3. DNA Methylation: Possible Target for Studies on Cancer Epigenetic Diagnosis & Therapy, 2015, Vol. 1, No. 1 7 inhibition of various miRNAs, tumor suppressor and functional genes [23, 24]. Moreover, it has become evident that different pathways target DNMT at the specific CpG Island. Moreover, four different mechanisms target DNA methyltransferase. Firstly, DNMT3A and DNMT3L recognize DNA or chromatin with specific domains and they interact with the LYS4 of histone 3 (H3K4). Secondly, DNMT interacts with different chromatin modifiers such as KMT and HDACs. Thirdly, the Piwi-PiRNA complex, a class of ncRNAs, guides DNMTs to transposon elements. Finally, the direct interaction of promoter-associated RNA (pRNA) with the promoter of the rRNA gene might recruit DNMT3b and enable methylation [25]. In addition, it has been shown that, in an alteration between methylation and phosphorylation, an adjacent lysine and serine determine the stability of DNMT1 thus preserving the methylation pattern during cell division [26]. However, the detailed mechanism(s) leading to hyper- or hypomethylation shall be decorticated in the future. HYPERMETHYLATION IN CANCER The CpG islands are protected by various defense mechanisms by accessing the DNA methyltransferase, such as DNA replication timing, active demethylation, or active transcription. Most genes that are susceptible to hypermethylation are associated with the regulation of DNA repair, apoptosis, cell cycle, drug resistance, metastasis, and differentiation [10]. Many tumors are hypermethylated in one or more genes. For instance, in lung cancer, it has been demonstrated that alterations of DNA methylation have been found in more than 40 genes [27]. Regarding breast cancer, it has been shown that several critical genes, which seem to be important in tumorigenesis, are hypermethylated. Among those genes, cell adhesion genes, inhibitors of matrix metalloproteinases, as well as the ER and PR steroid receptor genes can be found and have been linked to breast cancer development [28]. It has also been reported that DNA methylation is one of the reasons for the inactivation of BRCA1 in breast cancer, which is one of the genes most commonly associated with breast cancer [29]. Moreover, the hypermethylation of other cell cycle genes may result in acute lymphoblastic leukemia which is an undesirable clinical outcome [30]. HYPOMETHYLATION IN MALIGNANCIES Hypomethylation is an alteration in DNA methylation which is frequently seen in solid tumors [31-33], and is a sign of a progressive malignancy [34]. Furthermore, it has been reported that the hypomethylation status contributes to oncogenesis through up-regulation of some oncogenes, like H-RAs and c-MYC [31], as well as by chromosome instability [35, 36]. During cancer development, aberrations not only occur in the DNA methylation, but also in the regulation of histone modifications [37]. For instance, numerous studies have demonstrated that histone deacytylases (HDACs) are overexpressed in different types of cancer. This overexpression leads to a deacetylation of the Transcription Starting Site (TSS) of important genes and subsequently to the assembly of silenced genes into more compact structures [38, 39]. Fig. (2). Active demethylation pathway through the conversion of 5mC to 5hmC by recruiting TET proteins. Following 5mC oxidation, demethylation occurs by dilution of oxidized base or by removal through DNA glycosidase- and DNA repair-mediated excision of modified nucleotides (modified from [15]). Abbreviations: C: Cytosine, 5mC: 5-methylcytosine, 5hmC: 5-hydroxymethylcytosine; 5fC: 5- formylcytosine, 5caC: 5-carboxylcytosine, TDG: Thymine DNA Glucosidase, BER: Base Excision Repair.
  4. 4. 8 Epigenetic Diagnosis & Therapy, 2015, Vol. 1, No. 1 Sajadian and Nussler In summary, there exists an epigenetic crosstalk between histone modification, micro-RNAs, and DNA methylation. Post-transcriptionally, miR-29 and miR-148 regulate the DNA demethylases [40, 41]. Moreover, it has been proven that DNMTs interact with HDAC [28]. In particular, the combination of DNMT and HDAC inhibitors is a primary target for potential therapeutics against cancer [42, 43], because these inhibitors are capable of targeting the epigenetic status of DNA and histones simultaneously, which may enable the development of more effective anticancer drugs. EPIGENETICS AND CANCER STEM CELL HYPOTHESIS Recently, the concept has arisen that cancer stem cells are to be classified as a subpopulation of cancer cells with a different tumorigenic potential. This side population, which is usually named cancer-initiating cells or cancer stem cells (CSC), has a characteristic potential for self-renewal that may sustain and perpetuate tumors and is known to dispose of a higher drug resistance. Firstly, it has been observed that CD34+CD38- leukemic stem cells disposing of a differentiation and proliferation capacity are able to trigger human leukemia in NOD/SCID mice [44]. Furthermore, it has been proven that a landscape of solid tumors, such as glioblastoma [45], breast cancer [46], prostate cancer [47], and hepatocellular carcinoma [48], contain CSCs. A growing number of studies prove that the cancer-initiating cells are mostly resistant to chemotherapy or radiotherapy, which results in the recurrence of tumors [49], and in the formation of metastases [50]. Studies on the epigenetic modifications in stem cells and in cancer cells have directed interest to stem/progenitor cells, which are at the origin of cancer. It is believed that the genetic and epigenetic changes in these cells provide the ability for their survival, protecting them from toxic environment, which then contributes to tumor initiation [51]. Interestingly, the Polycomb repressive complex, which causes abnormal gene silencing in cancer, represses CpG island genes in Emberyonic Stem Cells (ESCs) in the same way as in cancer [52, 53]. Following this abnormal hypermethylation due to DNA damage, inflammation or stress caused by ROS, tumor suppressor genes become silenced. This may lead to the activation of stem cell pathways and provide the ability of self-renewal for cancer cells [54]. For example, in stem cells, like e.g. the CD133+ cells, promoter hypermethylation of some tumor suppressor genes, such as SFRP1 and SOX17, was identified [55], proving the stem cell origin of epigenetic abnormalities in cancer [56]. APPLICATION OF EPIGENETIC DRUGS As was described above, the application of epigenetic drugs is being increasingly focused on in the field of biomedicine and drug development research. Like numerous other diseases, cancer causes aberrancies in the epigenetic regulation. Most importantly, the pattern of DNA demethylation and histone modification has been related to tumor genesis and metastasis [10, 57]. Therefore, it is believed that these two biomechanical processes have a high potential for being used in clinical applications, such as e.g. in cancer detection, tumor prognosis, and pharmacoepigenetics (prediction of treatment response). For example, hypermethylation of glutathione transferase (GSTP1) constitutes a promising epigenetic marker in prostate cancer [58]. It has to be pointed out that 80-90% of men suffering from prostate cancer exhibit a hypermethylation of GSTP1 and that this hypermethylation does not occur in benign hyperplastic prostate tissue [59]. Therefore, GSTP1 methylation is a helpful prognostic marker, which enables the discrimination of benign tumor tissue in prostate cancer. As a matter of fact, GSTP1 is also hypermethylated in other cancers such as in kindney, liver and breast cancer [60]. Interestingly, the screening of single markers through global DNA methylation offers great clinical potential, as for example, it allows for the prediction of metastases in colorectal cancer, recurrence in lung cancer, and progression in virus-associated neoplasms [57, 61]. In addition to DNA methylation markers, histone-based markers have been suggested as epigenetic biomarkers. In particular, aberrant histone modifications have been linked to recurring prostate cancer [62]. Nowadays, most studies focus on the development of epigenetic drugs, which can reverse the abnormal epigenetic state to the normal epigenetic state in cancer cells. Up to now, the US Food and Drug Administration (FDA) has approved four epigenetic small molecule drugs for the treatment of cancer: 5-Azacytidine (5-Aza) and 5-Aza-2´- deoxycytidine (DNMT inhibitors) which have been used for patients suffering from myelodyplastic syndrome and from acute leukemia, as well as Varinostat and Romidepsin (HDAC inhibitors) which are used for treatment of T-cell lymphoma and some hematological tumors [63]. It has to be pointed out that each DNMT inhibitor possesses its own way of action on the gene transcription. Although 5-Aza and 5-Aza-2´-deoxycytidine have the same therapeutic effect, their mode of action in tumor cells is different. It has been demonstrated that 5-Aza-2´- deoxycytidine is able to induce senescence in tumor cells, while 5-Aza induces cell death in tumor cells in vitro and in vivo. These two DNMT inhibitors differ from each other on the molecular and the cellular level: For instance, 5-Aza can be integrated in both DNA and RNA (20-40% and 60-80% respectively), while 5-Aza-2´-deoxycytidine can only be incorporated in DNA. Moreover, 5-Aza-2´-deoxycytidine induces more DNA damage and micronuclei formation than 5-Aza [64]. Furthermore, it has been rported that Azacytidine increases the antitumor effect sensitivity of Docetaxel (DTX) and Cisplation in aggressive and chemoresistant prostate cancer [65]. In addition, it has been shown that Pyrazofurin (PF), an inhibitor of orotidylate decarboxylase improved the anti-tumoral activity of 5-Aza in human leukemia cells [66]. The combination of PF and 5-Aza may provide a useful treatment for patients who do not respond to standard anti- leukemic therapies [67]. Furthermore, it has been proven that 5-Aza-2´-deoxycytidine inhibits telomerase activity and reactivates p16 and c-Myc in hepatocellular carcinoma cells (HCC), such as the MMC-7721 and the HepG2 cell line [68]. Other DNMT inhibitors, such as 5-fuoro-2´- deoxycytidine, in combination with other drugs are currently
  5. 5. DNA Methylation: Possible Target for Studies on Cancer Epigenetic Diagnosis & Therapy, 2015, Vol. 1, No. 1 9 undergoing clinical trials regarding the treatment of different types of cancers [69]. The DNA methyltransferase inhibitor Zebularin provoked the demethylation of tumor suppressor genes by inducing apoptosis, cell cycle regulation in HCC cell lines (Huh7, KMCH, and HepG2), and in vivo inhibition of tumor growth in a xenograft model [70]. It has been discussed if the expression of specific DNMTs might influence the differentiation status in Fig. (3). Epigenetic drugs for cancer therapy. It has been described that scores of small molecules participate in the fight against cancer cells by inducing changes in the epigenetic machineries. This figure illustrates some of the most important epigenetic drugs, which are classified in relation to their epigenetic target. Abbreviations: DNMT1: DNA Methyltransferase 1, HDAC: Histone Deacetylase, PcG: Polycomb Group, Me: Methylated, HAT: Histone Acetylase. Repressive complex Repressive complex DNMT1DNMT1 PcG SirtuinsSirtuins HDACHDAC PcG MeDNMT Inhibitors: Vidaza Decitabine HMT Inhibitors DZNep BIX-01294 Me Decitabine Zelbularine Procainamide HDAC Inhibitors:HDAC Inhibitors: SIRTSIRT BIX 01294 Chaetocin DNMT1DNMT1 HDACHDAC Me VPA TSA Vorinostat Panobinostat Belinostat VPA TSA Vorinostat Panobinostat Belinostat Inhibitors Sirtinol Cambinol Salermide Inhibitors Sirtinol Cambinol Salermide PcG SirtuinsSirtuins Transcription HATHAT TETsTETs factors/ coactivator Pol IIPol II 5-methylcytosine5 methylcytosine 5-hydroxymethylcytosine 5-formylcytosine 5-carboxylcytosine Histone acetylated
  6. 6. 10 Epigenetic Diagnosis & Therapy, 2015, Vol. 1, No. 1 Sajadian and Nussler developing and dedifferentiating cells. It has been shown that 5-Aza triggers differentiation in mesenchymal stem cells (MSCs) [8, 71, 72]. Furthermore, we have reported that pretreatment of fat-derived old MSCs with 5-Aza improves osteogenic differentiation by DNA demethylation. We have observed a reduction of 5hmC in Ad-MSCs from older donors (> 60 years) compared to young donors (< 45 years). After stimulation of cells with 5-Azacytidine the osteogenic potential of the cells improved. It has been observed that the increase of AP activity and matrix mineralization is associated with an inducement of the expression of 5hmC as well as with the expression of TET2 and TET3 [8]. Therefore, we have concluded that 5-Azacytidine induces the rejuvenating process of older Ad-MSCs (Fig. 4). Furthermore, it has been demonstrated in various studies that the treatment of human Adipose Stem Cells (ADSC) and human bone marrow MSCs with 20 µM 5-Azacytidine improves their hepatic differentiation [73-75]. In the same line of evidence, it has been shown that the incubation of human bone marrow MSCs with 1 µM TSA which had been pretreated with hepatogenic-stimulating agents, improves the hepatogenic differentiation of these cells [76]. Moreover, a combination of TSA and 5-Aza-dc has improved the differentiation of human CD34+ to CD34/ CD90 cells [77]. Regarding the influence of the epigenetic status on the reprogramming mechanism of the cells, it has been reported that vitamin C induces the active demethylation pathway through the induction TET enzyme activity in mouse embryonic stem cells (ES) [78]. In the same line of evidence, it has been demonstrated that TET2 facilitates the differentiation of hematopoietic stem cells [79]. Therefore, vitamin C can be used for reprogramming stem cells, as it induces TET-dependent demethylation. OTHER EPIGENETIC MODIFYING DRUGS In the past years, many anticancer drugs targeting the acetylation or methylation status of histones through different mechanisms have been developed, such as HDAC inhibitors class I, II, III (sirtuins), and IV, HATs, HMTs, HDMs, and various kinds of kinases. Based on their chemical properties, the HDACs are branched into the four Fig. (4). Reprogrammation and differentiation of cells through a DNA methylation pattern. Changes in the methylation status of cells may lead to differentiation either through passive demethylation [8, 72] by inhibition of DNMT or through active demethylation by inducing TET proteins to produce 5hmC [78, 79]. Abbreviations: 5mC: 5-methylcytosine 5hmC: 5-hydroxymethylcytosine; 5fC: 5-formylcytosine; 5caC: 5-carboxylcytosine; TDG: Thymine DNA Glucosidase; BER: Base Excision Repair, TET: Ten eleven translocation, DNMT: DNA Methyltransferase, SAH: S-adenosylhomocysteine, Hcy: Homocysteine SAM: S-Adenosylmethionin. Progenitor / stem Cells Differentiated Cells DNMT Inhibitors: 5-AZA stem Cells MethionineSAH5mC 5caC C Pol II Progenitor Cells HcySAM 5hm C 5fC 5caC Pol IICells Differentiated Cells
  7. 7. DNA Methylation: Possible Target for Studies on Cancer Epigenetic Diagnosis & Therapy, 2015, Vol. 1, No. 1 11 following groups: hydroxamic acid, short fatty acids, cyclic peptide and benzamide enzymes [57]. HDACs inhibitors mainly arrest the cell cycle in G1 and G2-M phases, which, in turn, induces apoptosis and cell differentiation. However, they are able to inhibit angiogenesis and metastasis, and to decrease the resistance of tumor cells to chemotherapy [80]. Recently, a new epigenetic drug called Suberoylanilide hydroxamic acid (SAHA) has been approved for treating T- cell cutaneous lymphoma by the Federal Drug Administration (FDA) [81]. Furthermore, a large number of HDAC inhibitors, like e.g. TSA (Tricostatin A), PXD101 (Belinostat), and LBH589 (Panobinostat), are currently being developed and are undergoing clinical trials [82, 83]. Class III HDAC inhibitors, so called Sirtuins (SIRT), are important for the regulation of senescence, survival, and cell proliferation. An increasing number of effective SIRT inhibitors have recently been generated [57]. SIRT1 and SIRT2 inhibitors are likely to stop p53 activity, thereby inducing growth arrest in cancer cells and increasing sensitivity of tumor cells towards anticancer drugs [84]. Salermide - another SIRT1 and SIRT2 inhibitor - induces p- 53-independent apoptosis in tumor cells and reactivates proapoptotic genes by deacytelation of H4K16Ac at the epigenetic level which is mediated by SIRT1 [85]. It has been shown that curcumin, garcinol, and anacaridic acid may act as HAT inhibitors with an anti-cancer effect in ovarian and colon cancer. It has been demonstrated that all three substances are able to inhibit EP300-mediated p53 acetylation [86, 87]. DZNep, Chaetocin, and BIX-01294 are known to be lysine HMT inhibitors. It has been shown that DZNep promotes apoptosis in the MCF7 cell line (breast cancer) and in the HCT116 cell line (colorectal cancer) [88]. Chaetocine has an anti-cancer effect on multiple myeloma (MM) [89]. BIX-01294 deactivates the histone H3 lysine 9 (H3K9) methyltransferases G9a and G9a-like protein by promoting the apoptotic cell death [90]. It has been demonstrated that BIX-01294 induces endothelial differentiation in Ad-MSCs. We have been able to demonstrate that BIX modifies DNA and histone methylation and remarkably increases the expression of various endothelial markers that are required for blood vessel formation, such as VCAM-1, PECAM, VEGFR-2, and PDGF [72]. From our studies, we conclude that drugs that modify the DNA or histone methylation also induce changes in the pluripotency [8, 72]. SUMMARY AND FUTURE PERSPECTIVES In the present overview, we have presented numerous encouraging findings demonstrating that so called small molecules exert anti-tumoral activity through epigenetic changes within target cells. Interestingly, even molecules, like e.g. polyphenols, which are considered to be anti- oxidative drugs, dispose of a potent epigenetic modifying activity, and therefore slow down tumor growth. However, the large variety among the structures of different molecules that exert epigenetic modifying activity bears a high risk. The mechanisms by which epigenetically modifying drugs reduce growth or damage tumor cells are not very specific and may therefore damage normal/healthy cells as well. In this avenue, it is mandatory to develop new and more specific small molecules against specific enzymes of the epigenetic machinery in order to reduce damage on healthy tissue. CONFLICT OF INTEREST The authors declare that they do not have any conflict of interest. ACKNOWLEDGEMENTS We like to thank Dr. Luc Koster for editing the manuscript. 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