Epigenetic modulators from “The Big Blue”: A treasure to fight against cancer

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Cancer Letters 351 (2014) 182–197

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Cancer Letters

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Mini-review

Epigenetic modulators from ‘‘The Big Blue’’: A treasure to fight againstcancer

http://dx.doi.org/10.1016/j.canlet.2014.06.0050304-3835/� 2014 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: 5hmC, 5-hydroxymethylcytosine; 5mC, 5-methylcytosine; AID, activation-induced deaminase; AML, acute myeloid leukemia; APOBEC, apolipopmRNA editing enzyme, catalytic polypeptide-like; BAK, BCL2-antagonist/killer 1; BCL-2, B-cell lymphoma 2; BER, base excision repair; CBP, CREB-binding protecyclin-dependent kinase; CLDN4, claudin 4; CREB, cAMP response element-binding protein; DNMT, DNA methyltransferase; DNMTi, DNMT inhibitor; EGCG, epigallogallate; EHMT1, euchromatic histone-lysine N-methyltransferase 1; FAD, flavin adenine dinucleotide; FDA, food and drug administration; FOXO, forkhead box Ogeneral control nonderepressible 5; GNAT, GCN5-related N-acetyltransferase; GSTP, glutathione S-transferase pi; HAT, histone acetyltransferase; HDAC, histone deaHDACi, HDAC inhibitor; HDM, histone demethylase; HMT, histone methyltransferase; HOTTIP, HOXA transcript at the distal tip; HPLC, high-performancchromatography; IGF2, insulin-like growth factor 2; jmj, jumonji; L3MBTL, lethal 3 MBT-like protein; LINE, long interspersed element; lincRNA, long intergenic noRNA; lncRNA, long non-coding RNA; MAEL, mouse maelstrom; MBD, methyl CpG binding protein; MBT, malignant brain tumor; MCL-1, myeloid cell leukemia seqmicro-RNA, miRNA; MLH1, mutL homolog 1; MLL, mixed lineage leukemia; MOZ, monocytic leukemia zinc finger protein; MYST, MOZ, Ybf2/Sas3, Sas2, Tip6nicotinamide adenine dinucleotide; ncRNA, non-coding RNA; NF-jB, nuclear factor-kappa B; NMR, nuclear magnetic resonance; PCAF, p300/CBP-associated factorperoxisome proliferator-activated receptor gamma coactivator 1-alpha; PHD, plant homeodomain; PI3K, phosphatidylinositol 3 kinase; PRMT, protein argmethyltransferase; PsA, psammaplin A; PTEN, phosphatase and tensin homolog; PWWP, Pro-Trp-Trp-Pro; RAR, retinoic acid receptor; SAHA, suberoylanilide hydacid; SAM, S-adenosyl-methionine; SAR, structure–activity relationship; Sas, something about silencing protein; Sir2p, silent information regulator 2 protein; SIRTSTAT, signal transducers and activators of transcription; TET, ten-eleven translocation; Tip60, TAT-interacting protein 60; TSA, trichostatin A; TSG, tumor suppressUDG, uracil-DNA glycosylase; VPA, valproic acid.⇑ Corresponding author. Address: Department of Pharmacy, College of Pharmacy, Seoul National University, Building 20, Room 303, 1 Gwanak-ro, Gwanak-gu, Se

742, Republic of Korea. Tel.: +82 2 880 8919; fax: +82 2 880 2490.E-mail address: [email protected] (M. Diederich).

Michael Schnekenburger a, Mario Dicato a, Marc Diederich b,⇑a Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital Kirchberg, 9, rue Edward Steichen, L-2540 Luxembourg, Luxembourgb College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 19 March 2014Received in revised form 1 June 2014Accepted 4 June 2014

Keywords:Natural marine compoundsEpigeneticDNA methylationHistone modificationsMicro-RNAsAnti-cancer drugs

Cancer remains a major public health problem in our society. The development of potent novel anti-cancer drugs selective for tumor cells is therefore still required. Deregulation of the epigenetic machineryincluding DNA methylation, histone modifications and non-coding RNAs is a hallmark of cancer, whichprovides potential new therapeutic targets. Natural products or their derivatives represent a major classof anti-cancer drugs in the arsenal available to the clinician. However, regarding epigenetically activeanti-cancer agents for clinics, the oceans represent a largely untapped resource. This review focuses onmarine natural compounds with epigenetic activities and their synthetic derivatives displaying anti-cancer properties including largazole, psammaplins, trichostatins and azumamides.

� 2014 Elsevier Ireland Ltd. All rights reserved.

Introduction

Despite considerable progress in research and medicine, theburden of cancer in our society remains a major public healthproblem. Indeed, neoplastic diseases are a leading cause of deathwith an increasing incidence and mortality worldwide with lung,stomach, liver, colon and breast cancer causing the most cancerdeaths each year. Accordingly, cancer-related deaths are predictedto continuously increase, with an estimated 13.1 million deaths

worldwide in 2030 (source World Health Organization: http://www.who.int/mediacentre/factsheets/fs297/en/).

In this context, the identification of novel chemical entitiesallowing the development of potential novel anti-cancer agentsselective for tumor cells remains of major significance to extendthe therapeutic armamentarium for successful anti-cancer therapy.The therapeutic arsenal routinely use by oncologists rely on drugdiscovery of chemotherapeutic drugs from natural sources eitherunmodified or synthetically modified forms, which for most of

rotein Bin; CDK,catechin; GCN5,

cetylase;e liquid

n-codinguence 1;0; NAD,

; PGC1a,inine N-roxamic, sirtuin;or gene;

oul 151-

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M. Schnekenburger et al. / Cancer Letters 351 (2014) 182–197 183

them are from micro-organisms and plants with terrestrial origins.For instance, plant-derived compounds are widely used as anti-tumor agents such a vincristine and vinblastine from Catharanthusroseus, paclitaxel from Taxus brevifolia, etoposide from Podophyllumpeltatum while bleomycin, daunomycin and doxorubicin are bacte-rial products [32]. Nonetheless, oceans, covering over 70% of theEarth’s surface, contain an extensive biodiversity due to a tremen-dous variety of habitats housing marine plants, animals, andmicro-organisms well adapted to their environment. Oceans repre-sent undoubtedly a unique cradle of pharmacologically active newchemical structures, whose clinical potential remains largely unex-plored. Recently, marine compounds were shown to regulate mosthallmarks and enabling characteristics of cancer [140]. In particu-lar, related to cancer drug discovery cell death [47] and inflamma-tory [46,49,51] cell signaling pathways were studied in detail.Interestingly, photosynthetic marine organisms account as impor-tant sources for compounds with anti-cancer activity [48].

Accumulating evidence clearly established that, besidesgenetic lesions, epigenetic alterations including DNA methylation,histone modifications and micro-RNAs (miRNAs) are driving allsteps of carcinogenesis and play a critical role in initiation,development and maintenance of the tumor phenotype

Table 1Selected epigenetic drugs under clinical evaluation in cancer. The information related to clia compound is from natural origin, the common source is indicated.

Targetedactivity

Class ofcompound

Name Comments

DNMT Nucleosidesanalogues

5-aza-20-deoxycytidine(Decitabine, Dacogen�)

FDA-approved for mDNMT1

Azacytidine (Vidaza�) FDA-approved for mblocks DNMT1

SG-110 5-aza-20-deoxycytidNatural Curcumin From curcuma; decr

EGCG From green tea; decGenistein From soy; decreasesLuteolin From parsley, celery

Smallmolecule

Hydralazine Initially used in the

HAT Natural Curcumin From curcuma; inhiQuercetin From onion, broccol

HDAC Benzamides CI-994 (Tacedinaline) Inhibits HDAC1 andMGCD0103 (Mocetinostat) Inhibits HDAC1, 2, 3MS-275 (Entinostat) Inhibits class I HDA

Cyclic peptides None in clinical trialDepsipepides FK228 (Romidepsin) FDA-approved; fromHydroxamates CHR-3996 Inhibits class I HDA

ITF2357 (Givinostat) Inhibits class I and IJNJ-16241199 (R306465) Inhibits class I HDAJNJ-26481585 (Quisinostat) Inhibits class I and ILBH-589 (Panobinostat) Inhibits non-sirtuinNVP-LAQ824 (Dacinostat) Inhibits class I and IPCI-24781 (CRA-024781) Inhibits class I and IPXD101 (Belinostat) Inhibits non-sirtuinSAHA (Vorinostat) FDA-approved, inhibSB939 Inhibits non-sirtuin

Short-chainfatty acids

AN-9 (Pivanex,pivaloyloxymethyl butyrate)

Inhibits Class I, IIa a

Butyrate From gut fermentatSodium 4-phenylbutyrate Inhibits HDACsVPA Inhibits Class I and

Others 3,3-Diindolylmethane Digestive product oHDAC activity, down

CUDC-101 Inhibits HDACsGenistein From soy; inhibits nPhenethyl isothiocyanate From cruciferous veResveratrol From grape; sirtuinSuramin Inhibits SIRT1, 2 and

HMT E7438 Inhibits EZH2

DNMT: DNA methyltransferase, EGCG: epigallocatechin gallate, HAT: histone acetyltransirtuin, VPA: valproic acid.

[45,80,135,137,138]. The term ‘epigenetics’ refers to changes ingene expression independent of DNA sequence alterations. DNAmethylation, histone modifications and small RNA-mediated genesilencing are tightly associated mechanisms involved in the regu-lation of gene expression [91]. Noteworthy, unlike genetic modi-fications, epigenetic ones are potentially reversible; this specificfeature make epigenetic effectors an attractive target for anti-cancer treatments.

Over the past years, epigenetic drugs (i.e. compounds targetingepigenetic mechanisms) discovery has expended very rapidly andmany structures were identified as epigenetically active agents;however, only a restricted number of molecules have been clini-cally approved as epigenetic antineoplastic drugs, and a relativelysmall number are undergoing clinical trials (Table 1) [141]. In thiscontext, epigenetic drug research is seeking for novel lead struc-tures to develop more effective therapeutical agents. Consideringthat the marine ecosystem represents a largely unexplored sourceof molecular scaffolds; marine micro-organisms, plants, and ani-mals recently emerged as a promising resource of potent bioactivedrug candidates for anti-cancer therapy. Thus, in this review, wefocus on epigenetically active marine-derived compounds display-ing anti-cancer activities.

nical trials were retrieved from the following web site: http://clinicaltrials.gov/. When

References

yelodysplastic syndromes; incorporated into DNA and blocks [78]

yelodysplastic syndromes; incorporated into DNA (and RNA) and [90]

ine pro-drug [153]eases DNMT1 expression [101]reases DNMT expression [41]DNMT activity and expression [1]

; DNMT inhibitor [40]treatment of hypertension, DNMT inhibitor [31]

bits HAT (p300) [28]i, berries; sirtuin activator [71]2 [92]and 11 [52]

Cs [134]

the bacteria Chromobacterium Violaceum; inhibits class I HDACs [54]Cs [8]I HDACs [53]Cs [6]I HDACs [158]HDACs [129]I HDACs [21]Ib HDACs [20]HDACs [126]its non-sirtuin HDACs [128]HDACs [131]nd IV [38]

ion of dietary fibers; inhibits Class I, IIa and IV [33][123]

IIa [65]f indole-3-carbinol found in cruciferous vegetables; inhibits total

regulation of class I HDACs[13]

[93]on-sirtuin HDACs and increases HAT activity [103]getables; inhibits non-sirtuin HDACs [162]activator [42]

5 [154][88]

sferase, HDAC: histone deacetylase, SAHA: suberoylanilide hydroxamic acid, SIRT:

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Fig. 1. Overview of marine-derived compounds targeting epigenetic alterations involved in tumor suppressor gene silencing in cancer. Epigenetic alterations by silencing theexpression and therefore the functions of tumor suppressor genes contribute to carcinogenesis. These epimutations include increased promoter methylation in CpG islandregions of gene promoters associated with enrichment of histone repressive marks such as methylated H3K9 and H3K27 and a decrease of active histone marks includinghistone acetylated H3 and H4 and methylated H3K4. These modifications are mediated by the concert action of several enzymatic activities including: DNA methyltransferase(DNMT), histone acetyltransferase (HAT), sirtuin (SIRT) and non-sirtuin histone (HDAC), histone demethylase (HDM) and histone methyltransferase (HMT) activities.Epigenetically active marine-derived compounds are reported.

184 M. Schnekenburger et al. / Cancer Letters 351 (2014) 182–197

Epigenetic alterations in cancer

Epigenetic marks by regulating chromatin structure and tran-scription can switch genes on and off and control which ones aretranscribed to control cellular functions (Fig. 1). Alteration ofDNA methylation patterns was the first epigenetic mark to betightly link with carcinogenesis and to be responsible of alteredgene expression by the mean of tumor suppressor gene (TSG)silencing [43,135].

DNA methylation in cancer

In human, DNA methylation consists in the covalent addition ofa methyl group on cytosine residues (5’-position of the pyrimidinering) within CpG dinucleotides, resulting in the formation of5-methylcytosines (5mC). The reaction is catalyzed by the familyof DNA methyl transferases (DNMTs) using the cofactorS-adenosyl-methionine (SAM) as a methyl donor. Among this fam-ily, DNMT1 is denoted as the maintenance DNMT, as it owns thecapability to conserve methylation patterns through DNA replica-tion, due to its preference to hemi-methylated substrates. DNMT3Aand 3B are mainly involved in de novo DNA methylation. Therefore,these enzymatic activities act in concert to establish and maintainheritable genomic methylation patterns [39].

So far, there is no evidence for the existence of a DNA demeth-ylase activity that could catalyze DNA demethylation in a one-stepreaction. However, DNA repair and oxidative pathways linked tothe particular and unique chemical biology of cytosine could resultin DNA demethylation [109]. This pathway is controlled by ten-ele-ven translocation (TET) and activation-induced deaminase (AID)/apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like(APOBEC) – family of cytidine deaminases (see for review[16,150]). TET1, 2, 3 are 2-oxoglutarate- and Fe(II)-dependentenzymes capable of converting 5-methylcytosine (5mC) to5-hydroxymethylcytosine (5hmC), and then further to 5-formylcy-tosine and 5-carboxylcytosine by successive oxidative steps. 5mCand 5hmC are deaminated by the AID/APOBEC family members

to generate 5-methyluracil and 5-hydroxymethyluracil, respec-tively. These various cytosine derivatives are further targeted bythe uracil-DNA glycosylase (UDG) family of base excision repair(BER) glycosylases leading to cytosine replacement and DNAdemethylation.

In cancer cells, the alteration affecting DNA methylation themost thoroughly investigated is DNA hypermethylation. This epi-mutation corresponds to increased methylation at specific CpGdinucleotides that are usually unmethylated in healthy individuals.DNA hypermethylation occurs mainly at CpG islands, which corre-spond to GC-rich DNA regions of 200 bp to 4 kb-length with a GCcontent of at least 55% and a ratio of observed/statisticallyexpected CG frequencies of greater than 0.6. CpG islands are foundin about 60% of all genes, near promoter and exogenic regions[127]. Remarkably, a majority of cancer subtypes exhibit DNAhypermethylation at specific TSGs leading to their transcriptionalrepression. Examples of genes silenced by promoter hypermethy-lation include mutL homolog 1 (MLH1), retinoic acid receptor(RAR)-b and cyclin-dependent kinase (CDK) inhibitor genes(p16CDKN2A and p15CDKN2B) that are frequently methylated acrossvarious tumor subtypes. Moreover, genes such as glutathioneS-transferase pi (GSTP)1, are more specifically silenced in certaincancer subtypes, progressive methylation being a hallmark ofprostate cancer [39,45,81].

In cancer, local DNA hypermethylation is associated to globalgenomic DNA hypomethylation of highly repeated DNA sequencessuch as long interspersed element (LINE)-1 and certain targetgenes such as claudin (CLDN)4, maelstrom (MAEL), long non-coding(lnc)RNA H19/insulin-like growth factor (IGF)2 responsible forchromosomal instability and oncogenic activation, respectively[36,138].

Histone modifications and cancer

In eukaryotes, DNA is packaged with histone and non-histoneproteins into a higher-order structure of chromatin. Histones aretargeted by multiple post-translational modifications including

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acetylation, methylation, phosphorylation, ubiquitylation, sumoy-lation, citrullination, ADP-ribosylation b-N-acetylglucosaminationor deamination. Altogether these modifications are regulatingchromatin structure and activity [91,136].

The best-characterized modifications within histone tails areacetylation of lysine and methylation of arginine and lysine resi-dues. The acetylation/deacetylation processes are governed by his-tone acetyl transferases (HATs) and histone deacetylases (HDACs),respectively. Similarly, methylation/demethylation reactions arecatalyzed by histone methyltransferase (HMTs) and histonedemethylases (HDMs), respectively. Noteworthy, acetylation existsonly as a single addition, while multiple methylation levels (i.e.mono-, di-, and tri-methylation) can occur on the same residue[61].

HDAC isoenzymes are subdivided in four classes. Class I is com-posed of HDAC1, 2, 3 and 8, that are mainly located in the nucleus.Class II is further subdivided in subclass IIa with HDAC4, 5, 7 and 9;and subclass IIb with HDAC6 and 10. HDAC11 belongs to class IV.Class III, also called sirtuins, contains seven members, SIRT1-7.All members of HDAC class I, II and IV are zinc-dependent enzymeswhereas, sirtuins use nicotinamide adenine dinucleotide (NAD)+ asa cofactor. All HATS use acetyl-coenzyme A as an acetyl donor andare classified in five families from which three have been exten-sively studied: the p300/CBP family, the Gcn5-related N-acetyl-transferase (GNAT) family including the general control (GC)N5and P300/CBP-associated factor (PCAF) and the MYST (for thefounding members of this family: MOZ, Ybf2/Sas3, Sas2 andTip60) family [58]. Notably, HDACs not only regulate the acetyla-tion status of histone but also a myriad of other proteins. Forinstance, HDAC6 and SIRT2 target a-tubulin regulating microtu-bule stability [73]. The stability, subcellular localization andtranscriptional activity of many transcription factors (e.g. GATA,NF-jB, STAT) are also regulated by acetylation [50,142]. Interest-ingly, SIRT1/2 regulate the acetylation of additional proteins suchas p53, an important regulator of apoptosis, DNA repair, cell cyclearrest and metabolism [73]; forkhead box (FOX)O transcriptionfactors, key regulators of cell fate [94]; and peroxisome prolifera-tor-activated receptor gamma coactivator 1-alpha (PGC1a), a tran-scriptional co-activator that regulates mitochondrial biogenesisand functions [157].

The HMT family comprises a large number of enzymes usingSAM as a cofactor, subdivided in SET-domain-containing proteinsand DOT1-like proteins that target lysines, and protein arginineN-methyltransferase (PRMT) that target arginines (see for review[61]). HDMs were more recently identified and represent also alarge group of enzymes subdivided in two classes: the flavin ade-nine dinucleotide (FAD)-dependent monoamine oxidase familyand the both iron- and a-ketoglutarate-dependent dioxygenasejumonji (jmj) C-domain-containing proteins (see for review [61]).Similarly to HDACs/HATs, HDMs and HMTs target histone andnon-histone proteins.

Besides these writers and erasers of the epigenetic code, a thirdcategory of proteins emerged and plays an increasingly importantrole in epigenetically controlled gene expression. These proteinsare termed readers of the epigenetic code without having thecapacity to modify post-translation modifications of epigenetic tar-gets. Accordingly, compound regulators of bromodomain-contain-ing proteins, methyl-lysine- and/or methyl-arginine-bindingdomain-containing proteins including tudor domains, malignantbrain tumor (MBT) domains, chromodomains and Pro-Trp-Trp-Pro motif (PWWP) domains as well as plant homeodomain(PHD)-containing proteins become interesting targets for pharma-ceutically active compounds [5].

Altogether, the activity of these seven families of enzymes reg-ulates gene expression and therefore is implicated in many cellularprocesses. Consequently, it is well founded that alterations of

histone modifications patterns or the altered assessment of theepigenetic code by regulatory ‘‘readers proteins’’ are tightly associ-ated to tumorigenesis [34,125] and thus future druggable targets.

miRNAs in cancer

miRNAs are small non-coding (nc)RNAs of 19–25 nucleotides inlength that post-transcriptionally regulate mRNA expression lev-els. miRNAs bind sequences located essentially in 50 and 30

untranslated regions of target genes degrading mRNA or blockingtranslation [80]. miRNAs are probably targeting more than half ofall coding genes and largely contribute to regulating gene expres-sion. A growing body of evidence suggests that deregulation ofmiRNA expression patterns is associated to tumor development[45,80,125]. These altered miRNA expression profiles may resultfrom alterations of other epigenetic marks targeting miRNA genesor mutations affecting proteins involved in miRNA biogenesis andprocessing [80,125]. Besides small regulatory RNAs, lncRNAsattract increasing interest for anti-cancer therapy. LncRNAs are alarge and diverse class of transcribed RNA molecules with a lengthof more than 200 nucleotides that do not encode proteins. Thisfamily includes intronic as well as intergenic ncRNAs (lincRNAs).Although their role is still under investigation, removal of thesencRNAs is often associated with functional consequences. Remark-ably, lncRNAs interact with proteins as well as with RNA or DNA[108]. Conversely, lncRNAs are tightly coordinated to produce anintegrated regulatory effect and are emerging as master regulatorsof chromatin states by coordinating the role of writers, erasers andreaders. For instance, the lincRNA HOXA transcript at the distal tip(HOTTIP) modulates the activity of the WDR5-MLL complex, inwhich the WD40-repeat protein WDR5 binds the mixed-lineage,leukemia (MLL) complex to activate its H3K4 HMT activity leadingto the subsequent activation of targeted genes [35]. Another exam-ple demonstrating that lncRNA regulates chromatin remodelingand gene transcription is represented by the phosphatase and ten-sin homolog (PTEN) pseudogen PTENP1, which encodes two anti-sense RNA transcripts, a and b. The a isoform can recruitDNMT3A, the polycomb group protein EZH2 and the HMT G9A toPTEN promoter and repress its transcription [35]. This is just fewexamples of how lncRNAs can regulate epigenetic mechanismsand transcription (for review see [35,96]). Accordingly, deregula-tion of lncRNA expression profiles appears to be involved in tumor-igenesis and may account as future druggable targets [108].

Anti-cancer epigenetic drugs

Deregulation of the epigenetic machinery and aberrant epige-netic marks are together a common hallmark of cancer. These find-ings have been used as a driving force for the development ofpharmacological inhibitors for epigenetic-based anti-cancer thera-pies (Table 1).

In this context two nucleosides analogues able to inhibit DNMTactivities (DNMTi), 5-azacytidine and 20-deoxy-5-azacytidine, wereapproved by the FDA for the treatment of myelodysplasticsyndromes. Treatments with such molecules can lead to TSGre-expression associated with cycle perturbation, induction ofsenescence, autophagy, differentiation and apoptosis leading toimpaired cell proliferation and tumor regression [23,45,139,141].The success of these two molecules prompted researchers to gen-erate new derivatives with a better pharmacokinetic profile. Manymolecules have been generated but so far only SG-110 is undergo-ing clinical evaluation [62,141]. Non-nucleoside DNMTi may repre-sent a less toxic alternative to DNMTi that get incorporated intoDNA. This class includes both synthetic and naturally occurringmolecules; however, they usually display low potency and/or

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selectivity for DNMTs, and their mechanisms of inhibition are usu-ally not fully understood [62,141].

Among chromatin modifying enzymes, HDACs have receivedthe most attention as anti-cancer targets. Accordingly, severaldozen of naturally occurring and synthetic HDAC inhibitors(HDACi) have been reported to date, which include benzamides,cyclic peptides, depsipeptides, hydroxamates and short fatty acids.These agents share structure similarities allowing to defining aHDAC pharmacophore consisting of a metal-binding moiety thatinteracts with the catalytic zinc ion, a hydrophobic linker regionthat mimics the substrate’s lysine chain and a cap that blocks theaccess of the substrate to the active site. Two HDACi namely sube-roylanilide hydroxamic acid (SAHA, vorinostat, Zolinza�) andFK228 (Romidepsin, Istodax�) have been FDA-approved for thetreatment of cutaneous T-cell lymphoma. SAHA is a syntheticderivative of trichostatin A (TSA, see below 4.16.), which displayssimilar biological properties to its parent compound; however, itis a more potent pan-HDACi with less toxicity against normal cellsand side effects leading to its approval [29,141]. FK228 is so far theonly epigenetic drug from natural origin to receive FDA approval.This depsipeptide acts as a prodrug from which the disulfide bondis reduced in vivo to reveal butenylthiol, which is implicated inclass I HDAC inhibition. Despite potent activity against a broadrange of tumor cells, FK228 displays serious cardiac side effects,which may limit its clinical use [105,141].

Remarkably, besides FDA-approved compounds and the familyof HDACi there is very limited number of epigenetic drugs in clin-ical trials (Table 1).

Marine and marine-derived compounds with anti-canceractivities acting as epigenetic modulators

In the past few years, an increasing number of epigenetic mod-ulators with marine origins were reported (Fig. 1). So far, there isno HAT or HDM inhibitor of marine origin reported and the vastmajority of epigenetic modulators identified are HDAC inhibitors.Below we discuss marine-derived drugs with epigenetic activitiesdisplaying anti-cancer properties (Table 2 and Fig. 2).

1386A

1386A is a compound isolated from a mangrove fungus with thesame name collected in the South China Sea. This compound dis-plays cytotoxic effects (IC50 of 17.1 lM after 48 h of exposure) inhuman breast MCF-7 cancer cells leading to a time- and dose-dependent inhibition of cell growth [151]. Authors have shownthat MCF-7 cells exposed for 48 h to the IC50 concentration of1386A display 45 miRNAs differentially expressed. Target predic-tion revealed that altered miRNAs potentially target several onco-genes and TSGs linked to cancer development, progression andmetastasis that could explain the anti-cancer properties of thisdrug [151]. Further investigations are required to confirm thesedata and decipher the mechanism(s) of action of this new drug.

Azumamides

Nakao et al. were the first group to report the extraction, puri-fication, structure determination and biological evaluation of fivenew and unusual cyclic tetrapeptides, named azumamides A-E(1–5), isolated form the Japanese marine sponge Mycale izuensis[111]. These molecules consist of four non-ribosomal amino acidresidues, among which three are a-amino acids of the D-series(D-Ala, D-Phe, D-Val, D-Tyr), while the fourth one is a uniqueb-amino acid assigned as (Z)-(2S,3R)-3-amino-2-methyl-5-non-ene-dioic acid, 9-amide in azumamides A (1), B (2) and D (4),

and as (Z)-(2S,3R)-3-amino-2-methyl-5-nonen-dioic acid inazumamides C (3) and E (5) [74].

Azumamides A-E (1–5) were first identified as in vitro inhibitorsof total HDAC activity with IC50 values ranging from 0.045 to1.3 lM using human chronic myeloid leukemia K-562 crude celllysates. Accordingly, azumamide A (1) increases histone acetyla-tion in K-562 cells in a concentration-dependent manner from0.19 to 19 lM and induces cytotoxic effects in human colon cancerWiDr cells and K-562 cells with IC50 values of 5.8 and 4.5 lM,respectively [111]. Surprisingly, later it was demonstrated thatonly azumamides B (2), C (3) and E (5) were in vitro HDACi withIC50 values in the low micromolar range using HeLa (human cervixcarcinoma) nuclear extracts and class I HDACs (mainly the isoen-zymes 1 and 4) isolated from human embryonic kidney 293T cells.Furthermore, in these studies, derivative E (5) was the most potentcompound due to its carboxylic acid warhead [104,110,163], whichhas a better affinity for zinc than an amide group (found in azuma-mides A (1), B (2), D (4)) based on the model of pharmacophoreinhibition of non-sirtuin HDACs [141,142]. In another study, usingtwo concentrations of compounds (5 and 50 lM) and recombinanthuman HDACs, it was shown that azumamide C (3), bearing a car-boxylic acid moiety, was more potent against HDAC activities thanderivative E (5) [159]. These discrepancies might be explained bydifferences in compound purity in both natural and synthetic com-pounds as well as by the type of assay utilized.

Remarkably, azumamide E (5) is the only compound able topromote the expression of the CDK inhibitor p21 with an EC1000

value of 17.0 lM in mouse induced pluripotent stem cells [110];a well-accepted marker induced by HDACi, which is inhibiting cellcycle progression [141,142]. The anti-cancer effect of azuzamide A(1) was associated to anti-angiogenic properties at 19 lM using amouse in vitro model of vascular organization [111]. In anotherstudy, using the same experimental model, the same group dem-onstrated that azumamide E (5) displayed even more potentanti-angiogenic properties starting as low as 0.19 lM [110].

Various synthetic route of azumamides A (1) [74,159,163] and E(5) [22,74,159,163] were published, while so far only one syntheticroute of azumamides B-D (2–4) was reported [159]. These studieshave generated various analogues useful to assess azumamide SARtowards HDAC activities. These studies revealed that the b-aminoacid residue is essential for the HDAC inhibitory activity of azuma-mides. Among analogues two molecules displayed more potentHDAC inhibitory activities: azumamide E-SAA, a sugar amino acidderivative analogue, and one in which the carboxylic warhead isreplaced by a hydroxamic one, but these compounds were not fur-ther investigated in cells [22,163].

Bispyridinium alkaloids

Pyridine alkaloids are a group of nitrogen-containing secondarymetabolites very common in marine sponges, where they haveanti-microbial properties. Oku et al. identified a group of structur-ally related macrocyclic bispyridinium alkaloids called cyclostel-lettamines A (6) and G (7), and dehydrocyclostellettamine D (8)and E (9) able to inhibit HDAC activity from K-562 cells. Thesecyclostellettamines were isolated from a marine sponge of thegenus Xestospongia collected in Japan waters [117]. However, theirHDAC inhibitory activity is rather weak (IC50 = 17–80 lM) com-pared to their cytotoxic potential (IC50 = 0.6–11.0 lM) againsthuman cervix carcinoma HeLa, mouse leukemia P388 and rat fibro-blast 3Y1 cell lines [117]. This difference suggests the existence ofadditional target(s) of these compounds mediating their cytotoxicproperties.

Pérez-Balado et al. reported a synthetic route to natural cyclo-stellettamines A-L and dehydrocyclostellettamines D (9) and E(10) using as the key step a microwave-mediated macrocyclic

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Table 2Marine epigenetic modulators. The structures of the after mentioned compounds are reported in Fig. 2.

Compound Chemical class Source Epigenetic target(s) and mechanism(s) References

Organism Name Location

1386A Structure not available Fungus 1386A South China Sea Modulates miRNA expression profile in human breast cancer MCF-7 cells1: " miR-7, -21, -181b, -200c, -203, -638, -654-5p, -663, -1246, -1826 ; miR-25, -93, -125, -150, -182, -320family, -1308, -let7 family

[151]

Azumamide A (1) Cyclic peptide Sponge Mycale izuensis Japan waters In vitro2 HDACi, increases histone acetylation [104,110,111]Azumamide B (2) Cyclic peptide Sponge Mycale izuensis Japan waters In vitro HDACi, class I HDACi [104,110,111]Azumamide C (3) Cyclic peptide Sponge Mycale izuensis Japan waters In vitro HDACi, class I HDACi [104,110,111]Azumamide D (4) Cyclic peptide Sponge Mycale izuensis Japan waters In vitro HDACi [104,110,111]Azumamide E (5) Cyclic peptide Sponge Mycale izuensis Japan waters In vitro HDACi, class I HDACi, increases p21 expression [104,110,111]Cyclostellettamine A (6) Bispyridinium alkaloid Sponge Genus

Xestospongia(Haliclona sp.)

Shishijima Island, TheAmakusa Islands,Southern Japan

In vitro HDACi, increases histone acetylation [117,121]

Cyclostellettamine G (7) Bispyridinium alkaloid Sponge GenusXestospongia(Haliclona sp.)

Shishijima Island, TheAmakusa Islands,Southern Japan

In vitro HDACi [117]

DehydrocyclostellettamineD (8)

Bispyridinium alkaloid Sponge GenusXestospongia(Haliclona sp.)

Shishijima Island, TheAmakusa Islands,Southern Japan

In vitro HDACi [117]

DehydrocyclostellettamineE (9)

Bispyridinium alkaloid Sponge GenusXestospongia(Haliclona sp.)

Shishijima Island, TheAmakusa Islands,Southern Japan

In vitro HDACi [117]

Bostrycin (10) Anthraquinone Fungus Nigrospora sp.(No. 1403)

South China Sea Modulates miRNA expression profile inhuman lung cancer A-549 cells1: " miR-638 and -923 [26,27]

Gliotoxin G (15) Sulfur-containing toxin Fungus Penicillium sp.(strain JMF034)

Suruga Bay, Japan In vitro HMTi (G9a) [148]

5a,6-didehydrogliotoxin(12)

Sulfur-containing toxin Fungus Penicillium sp.(strain JMF034)

Suruga Bay, Japan In vitro HMTi (G9a) [148]

Gliotoxin (13) Sulfur-containing toxin Fungus Penicillium sp.(strain JMF034)

Suruga Bay, Japan In vitro HMTi (G9a) [148]

Gymnochrome E (14) Phenanthroperylenequinone Crinoid Holopus rangii South coast of Curacao In vitro HDAC1i [82]Largazole (16) Depsipeptide Bacteria Genus Symploca Key Largo, Florida Keys,

USAIn vitro HDACi, class I HDACi, increases histone acetylation [15,17,68,169]

Microsporin A (17) Cyclic peptide Fungus Microsporumgypseum sp.

US Virgin islands [63]

Nahuoic acid A (18) Polyketide Bacteria Streptomycessp. (isolateRJA2928)

Padana Nahua passage,Papua New Guinea

In vitro HMTi (SETD8) [165]

Peyssonenyne A (19) Fatty acid Algae Peyssonneliacaulifera

Yanuca Island, Fiji,Melanesia

In vitro DNMTi [56,106]

Peyssonenyne B (20) Fatty acid Algae Peyssonneliacaulifera

Yanuca Island, Fiji,Melanesia

In vitro DNMTi [56,106]

Psammaplin A (21) 3-Bromotyrosine derivative Sponge Pseudoceratinapurpurea

Papua New Guinea andMadagascar

In vitro DNMTi, In vitro HDACi [124]

Bisaprasin 3-Bromotyrosine derivative Sponge Pseudoceratinapurpurea

Papua New Guinea andMadagascar

In vitro DNMTi, In vitro HDACi [124]

Psammaplin G (22) 3-Bromotyrosine derivative Sponge Pseudoceratinapurpurea

Papua New Guinea andMadagascar

In vitro DNMTi, In vitro HDACi [124]

Psammaplin B (23) 3-Bromotyrosine derivative Sponge Pseudoceratinapurpurea

Papua New Guinea andMadagascar

In vitro HDACi [124]

Psammaplin D (24) 3-Bromotyrosine derivative Sponge Pseudoceratinapurpurea

Papua New Guinea andMadagascar

In vitro HDACi [124]

Psammaplin E (25) 3-Bromotyrosine derivative Sponge Pseudoceratinapurpurea

Papua New Guinea andMadagascar

In vitro HDACi [124]

Psammaplin F (26) 3-Bromotyrosine derivative Sponge Pseudoceratinapurpurea

Papua New GuineaandMadagascar

In vitro HDACi [124]

Psammaplin H (27) 3-Bromotyrosine derivative Sponge Pseudoceratinapurpurea

Papua New Guinea andMadagascar

In vitro HDACi [124]

(continued on next page)

M.Schnekenburger

etal./Cancer

Letters351

(2014)182–

197187

Page 7

Tabl

e2

(con

tinu

ed)

Com

pou

nd

Ch

emic

alcl

ass

Sou

rce

Epig

enet

icta

rget

(s)

and

mec

han

ism

(s)

Ref

eren

ces

Org

anis

mN

ame

Loca

tion

Psam

map

lin

I(2

8)3-

Bro

mot

yros

ine

deri

vati

veSp

onge

Pseu

doce

rati

napu

rpur

eaPa

pua

New

Gu

inea

and

Mad

agas

car

Invi

tro

HD

AC

i[1

24]

Psam

map

lin

J(2

9)3-

Bro

mot

yros

ine

deri

vati

veSp

onge

Pseu

doce

rati

napu

rpur

eaPa

pua

New

Gu

inea

and

Mad

agas

car

Invi

tro

HD

AC

i[1

24]

San

tacr

uza

mat

eA

(31)

Cyt

otox

inB

acte

ria

Gen

us

Sym

ploc

asp

.C

oiba

Nat

ion

alPa

rk,

Pan

ama

Invi

tro

HD

AC

2i[1

19]

Stre

ptos

etin

A(3

2)B

acte

ria

Stre

ptom

yces

viol

aceu

snig

erSa

nFr

anci

sco

Bay

,USA

Invi

tro

SIR

T1i

and

SIR

T2i

[3]

SZ-6

85C

(33)

An

thra

quin

one

Fun

gus

Nig

rosp

ora

sp.

(No.

1403

)So

uth

Ch

ina

Sea

;m

iR-2

00c

[24]

Tan

ikol

ide

dim

er(3

4)D

ilac

ton

eB

acte

ria

Lyng

bya

maj

uscu

laTa

nik

ely

Isla

nd,

Mad

agas

car

Invi

tro

SIR

T1i

and

SIR

T2i

[66]

Tric

hos

tati

nA

(35)

Hyd

roxa

mat

eB

acte

ria

Stre

ptom

yces

angu

stm

ycin

icus

Taka

raIs

lan

d,Ja

pan

Invi

tro

HD

AC

i,n

on-s

irtu

inH

DA

Ci,

incr

ease

spr

otei

nac

etyl

atio

nan

din

crea

ses

p21

expr

essi

on[2

9,70

,170

]

Tric

hos

tati

cac

id(3

6)Tr

ich

osta

tin

anal

ogu

eB

acte

ria

Stre

ptom

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angu

stm

ycin

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Taka

raIs

lan

d,Ja

pan

Invi

tro

HD

AC

1i[7

0]

JBIR

-109

(37)

Tric

hos

tati

nan

alog

ue

Bac

teri

aSt

rept

omyc

esan

gust

myc

inic

usTa

kara

Isla

nd,

Japa

nIn

vitr

oH

DA

C1i

[70]

JBIR

-110

(38)

Tric

hos

tati

nan

alog

ue

Bac

teri

aSt

rept

omyc

esan

gust

myc

inic

usTa

kara

Isla

nd,

Japa

nIn

vitr

oH

DA

C1i

[70]

JBIR

-111

(39)

Tric

hos

tati

nan

alog

ue

Bac

teri

aSt

rept

omyc

esan

gust

myc

inic

usTa

kara

Isla

nd,

Japa

nIn

vitr

oH

DA

C1i

[70]

DN

MTi

:D

NA

met

hyl

tran

sfer

ase

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ibit

or,H

DA

Ci:

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ton

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acet

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in.

1Th

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up-

and

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n-r

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late

dm

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-RN

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(mi-

RN

As)

.2

Invi

tro

mea

ns

dete

rmin

edby

cell

-fre

ebi

och

emic

alas

say.

188 M. Schnekenburger et al. / Cancer Letters 351 (2014) 182–197

ring-closing metathesis of precursors bispyridinium dienes fol-lowed by catalytic hydrogenation [121]. For most compoundsand precursors, authors systematically investigated their effecton HDAC activity as well as p21 protein expression, histone H3and a-tubulin acetylation, cell cycle distribution, granulocytic dif-ferentiation, and apoptosis in human histiocytic lymphoma U-937cells using 1 or 5 lM of each compounds. Interestingly, acyclicbispyridinium diene precursors are globally more potent HDACiand display increased biological activities compared to the naturalcompounds. For instance, the open-chain precursor 10b is aHDAC1-selective inhibitor inducing histone H3 acetylation andp21 expression and 10a is a HDAC subclass IIa-selective inhibitorinducing G1 cell cycle accumulation without affecting protein acet-ylation. Nevertheless, all open-chain bispyridinium dienes induceapoptosis after 30 h of treatment at the concentration of 5 lM[121].

Bostrycin

Bostrycin (10) a natural tetrahydroanthraquinone pigmentisolated from several marine-derived fungus collected from themangrove of South China Sea: Nigrospora sp. (No. 1403), Xylariasp. (2508) [26,72]; and coral reef of Manado (Indonesia):Aspergillus sp. (strain 05F16) [168].

Bostrycin (10) displays cytotoxic properties in various humancancer models including lung A-549, breast MCF-7 and MDA-MB-435, liver HepG2, and colon HCT-116 cell lines with IC50 valuesbetween 2.2 and 7.7 lM after 48 h of treatment. Compared to theimmortalized human breast epithelial cell line MCF-10A, bostrycin(10) has a 2- to 7-fold selectivity against cancer cell lines [26,27].Furthermore, 10–30 lM of this compound inhibits proliferationand induces the expression of the CDK inhibitor p27, G0/G1 cellcycle arrest and apoptosis in human lung carcinoma A-549 cellsby down-regulating the PI3K/AKT pathway [27]. Finally, Chenet al. investigated miRNA expression profile in A-549 cells exposedto 10 lM bostrycin (10) for 72 h and found 54 mi-RNA differen-tially expressed with miR-638 and -923 being the most upregu-lated [27].

Gliotoxins

Several gliotoxins were isolated from the fungus Penicillium sp.strain JMF034, obtained from deep-sea sediments of Japan. Spec-troscopic data revealed five already known compounds and twonew metabolites. The known compounds were identified asbis(dethio)bis(methylthio)gliotoxin already characterized in thefungus Gliocladium deliquescens [87], bis(dethio)bis-(methylthio)-5a,6-didehydrogliotoxin and gliotoxin G (11) found initially inthe fungus Gliocladium virens [86], 5a,6-didehydrogliotoxin (12)isolated from the fungus Gliocladium flavofuscum [7], and gliotoxin(13) already characterized in the fungus Dichotomomyces cejpii[79]. The new compounds were identified as bis(dethio)-10a-methylthio-3a-deoxy-3,3a-didehydrogliotoxin and 6-deoxy-5a,6-didehydrogliotoxin [148]. Although all compounds exhibitedcytotoxic activity against P388 murine leukemia cells (IC50 valuesbetween 0.02 and 3.4 lM after 96 h of treatment), only compoundscontaining a disulfide bond, namely gliotoxin G (11), 5a,6-didehy-drogliotoxin (12) and gliotoxin (13) showed potent in vitro inhibi-tory activity against the recombinant H3K9 HMT G9a (IC50 =2.1–6.4 lM) without affecting the H3K4 HMT SET7/9 [148].

Gymnochrome E

Gymnochrome E (14) is a cytotoxic brominated phenanthro-perylenequinone pigments isolated from the deep-water crinoidHolopus rangii. Gymnochrome E (14) inhibits the proliferation of

Page 8

Fig. 2. Structures of natural marine-derived epigenetic modulators. The structure of compound 1386A reported in Table 2 is not available. *Synthetic compound.

Fig. 2 (continued)

M. Schnekenburger et al. / Cancer Letters 351 (2014) 182–197 189

the multi-drug-resistant ovarian NCI/ADR-Res cancer cell line(IC50 = 3.5 lM) but had no effect at a concentration of 6.4 lM onpancreatic carcinoma PANC-1 or human colorectal adenocarci-noma DLD-1 cell lines. Furthermore, gymnochrome E (14) alsoinhibits HDAC1 activity with an IC50 of 10.9 lM [82]. Remarkably,gymnochrome F (15), a co-purified and structurally related

compound to gymnochrome E (14), lacks HDAC inhibitory activityand significant toxicity against PANC-1, NCI/ADR-Res and DLD-1tumor cell lines at a concentration of 5.2 lM [82]. GymnochromeE (14) is also an inhibitor of the binding of the anti-apoptotic mem-ber of the BCL-2 family, MCL-1, to the pro-apoptotic BCL-2 memberBAK (IC50 = 3.3 lM), which regulates apoptosis once released [82].

Page 9

Fig. 2 (continued)

190 M. Schnekenburger et al. / Cancer Letters 351 (2014) 182–197

Largazole and its analogues

Largazole (16) is a highly functionalized macrocyclic depsipep-tide originally isolated from a cyanobacterium of the genusSymploca [152]. Largazole (16) was trivially named based on itslocation of collection, Key Largo (Florida Keys, USA), as well asthe presence two azole-like moieties: an a-methylcysteine-derivedthiazoline coupled to a thiazole. These characteristic chemicalfeatures are embedded in an unusual strained 16-memberedmacrocycle possessing a remarkable scaffold including a caprylicacid-derived thioester, which is rarely encountered in naturalproducts. The structure of largazole (16) contains also one non-modified L-valine amino acid [68,152].

Largazole (16) was initially revealed to display very potentgrowth-inhibitory potential against various cancer cell models(GI50: 7.7–55 nM), whereas non-transformed fibroblasts epithelialcells were inhibited at much higher doses (GI50: 122–480 nM).The selectivity of largazole (16) for cancer cells was unmatchedby other natural drugs tested including paclitaxel, actinomycin Dand doxorubicin [112,152]. Later these data were extended to theNational Cancer Institute’s 60 cell line system showing that largaz-ole (16) has a highly effective growth-inhibitory potential againstmany cell lines with a mean GI50 value of 17 nM (1.6–320 nM).Accordingly, largazole (16) displays cytotoxic effects at muchhigher concentrations (mean LD50 = 18.6 lM) [99].

The remarkable selectivity of largazole (16) against cancer cellsprompts the scientific chemistry community to establish syntheticroutes of this compound for extensive biological evaluation of itsmechanisms of action [14,15,17,18,59,112,116,132,143,147,160,166,169,173]. The different synthetic routes have been extensivelydiscussed in several reviews [68,97,114].

Largazole (16) is a depsipeptide with the intriguing presence ofa thioester functionality expected to be swiftly hydrolyzed throughcellular metabolism to generate the corresponding thiol form oflargazole as observed with three other naturally occurring depsi-peptides: FR901375, FK228, and spiruchostatin A, which are allwell-established HDACi [141]. Based on these features, Lueschet al. were not only the first group to propose a total synthesis oflargazole and some analogues including the thiol form, but alsothe first to demonstrate its HDAC inhibitory properties [169].Indeed, largazole inhibits both in vitro and in cellulo total HDACactivity (IC50 around 50 nM) and induces histone H3 acetylationin a concentration-dependent manner starting about 1–10 nMwithout affecting a-tubulin acetylation. These findings were con-firmed by latter studies that further demonstrate that largazole(16) displays class I selectivity profile [15,17,68,169].

A wide range of largazole analogues have been synthesized tomodulate its structural scaffold focusing on the thioester linkerregion, the L-valine subunit, and the 4-methylthiazoline–thiazolesubunit with the goal to deeply investigate the SAR of this com-pound [14,15,18,19,25,59,64,112,143,147,160,169,173]. Thesestudies demonstrated that largazole (16) acts as a prodrug andthe thiol form is the active metabolite, which is indispensable forboth activities (i.e. HDAC inhibition and anti-proliferation). Accord-ingly, in vitro assay showed that the thiol form is a much morepotent HDACi (IC50 in the picomolar range) [17]. Nonetheless, allthese studies failed to identify largazole analogues with a morepotent HDAC inhibitory activity than the parent compound. Unfor-tunately, several studies only investigated the effect of these deriv-atives on cell proliferation but not on HDAC activity or only onHDAC activity without testing the effect on protein acetylation.Nonetheless, altogether these studies suggest the importance of

Page 10

Fig. 2 (continued)

M. Schnekenburger et al. / Cancer Letters 351 (2014) 182–197 191

positioning the thiol functionality at the right distance from thecyclic core and the necessity of the thiobutenyl group for its anti-proliferative activity. Remarkably, L-valine can be replaced withL-alanine or glycine without drastic loss of HDAC inhibitory activity[14,169]. Furthermore, Cole et al. demonstrated that other L-aminoacids might be used at this position as long as they do not perturbthe overall conformation of the macrocyclic depsipeptide core [30].However, by exchanging L-valine by L-tyrosine in largazole (16)enhanced its selectivity toward human cancer cells versus normalcells over 100-fold but authors did not test the effect on HDACactivity or protein acetylation [173]. Interestingly, the analogue2-epi-largazole was less potent that largazole (16) on HDAC activ-ity but about two times more cytotoxic on PC-3 and LNCaP prostatecancer cell lines [160].

Several studies have investigated the downstream effects of lar-gazole (16) associated to decreased cancer cell proliferation. Inhuman colon HCT116 cancer cells low concentrations (1–3.2 nM)of largazole (16) cause G1 cell cycle arrest, whereas higher concen-trations (10 nM) lead to G2/M arrest. Higher concentrations(>10 nM) induce caspase activity and trigger apoptosis. Further-more, largazole (16) induces several CDK inhibitors (p15, p19,p21, p57) and down-regulates CDK6 and cyclin D1 in NB4 andHCT116 cells [99,147]. In vivo, using a HCT116 xenograft mousemodel, 5 mg/kg largazole (16) administrated by intraperitonealinjection induces protein acetylation and caspase activation lead-ing to a reduction of tumor growth and volume [99].

Recently it was demonstrated that 125 nM largazole (16) haveanti-fibrotic and anti-angiogenic properties both in vitro usinghepatic stellate cells (HSCs) and in vivo using a mouse liver fibroticmodel by inhibiting transforming growth factor-b and vascularendothelial growth factor signaling, while largazole (16) had noeffect on normal hepatocytes. Remarkably, these effects are depen-dent of the HDAC inhibitory activity of largazole (16) [100]. Furtherstudies demonstrate that 100 nM largazole up-regulates epigenet-ically-silence E cadherin gene and change its subcellular localiza-tion in breast cancers MDA-MB231 cells. These effects are

associated to a suppression of cell invasion in vitro and enhanceddexamethasone effects [95].

Besides its effect on HDAC activity, it was shown that largazole(16) acts as an ubiquitin E1 inhibitor (IC50 = 29 lM) that couldaccount for its anti-cancer properties [155].

Microsporin A

Microsporin A (17) is a cyclic tetrapeptide purified from the cul-ture of the marine-derived fungus Microsporum gypseum sp. iso-lated from a sample of the bryozoan Bugula sp. collected in theUS Virgin Islands [63]. This molecule is constituted of three aminoacids with a L configuration, while the pipecolic acid present a Dconfiguration.

Notably, authors have reported that microsporin A (17) pos-sesses a similar inhibitory activity than SAHA against in vitro totalHDAC activity (IC50 values of 0.14 and 0.30 lM for microsporin A(17) and SAHA, respectively) and displays cytotoxic propertiesagainst human colon HCT-116 cancer cells (IC50 = 1.2 lM) as wellas against the National Cancer Institute 60 cancer cell panel witha mean IC50 value of 2.7 lM [63]. These promising data require fur-ther investigations to determine whether the biological activity ofmicrosporin A (17) is related to its HDAC inhibitory potential.

Nahuoic acid A

Nahuoic acid A (18) is a polyketide produced in culture by abacteria Streptomyces sp. (isolate RJA2928) obtained from a tropicalmarine sediment near the Padana Nahua passage in Papua NewGuinea. Interestingly, nahuoic acid A (18) is the first selectiveSAM-competitive inhibitor of the HMT SETD8 activity (IC50 = 6.8lM) reported without affecting the activity of other HMTs suchG9a, EHMT1, SETD7, SUV39H2, SUV420H1, SUV420H2, DOT1L,PRMT3, and PRMT5 and MLL complexes [165]. SETD8 is a HMT thatplays a key role in the silencing of euchromatic genes by monom-ethylating histone H4 (lys 20) [61]. In addition, this enzymerepresses p53 target genes by methylating p53 [144] and enhancescell proliferation by methylating PCNA [149]. In this context, sinceSETD8 is found overexpressed in various cancers [149], it mightrepresent an interesting therapeutic target in cancer. Therefore, itwould be interesting to investigate the effect of nahuoic acid A(18) on cancer cells and to evaluate the effect of this structure onHDAC activities since other polyketides were reported as HDACi[37,164].

Peyssonenynes

Peyssonenynes A (19) and B (20) are x3 fatty acids possessingan unusual enediyne motif isolated from the Fijian red marinealgae Peyssonnelia caulifera. They were identified as DNMTi usinga bioassay-guided fractionation [106].

These compounds were initially reported to be geometric iso-mers at the acetoxyenediyne moiety; however, the full synthesisof these molecules coupled to extensive analysis of NMR and HPLCdata revealed that peyssonenynes A (19) and B (20) are the sn-1,3and sn-2 positional isomers at the glycerol moiety, respectively[56]. In the initial study, pure peyssonenynes A (19) and B (20)were shown to display comparable activities against purifiedDNMT1 enzyme in in vitro activity assay (IC50 values of 16 and9 lM, respectively) [106]. However, using DNMT1 immunoprecip-itated from K-562 cells, Garcia-Dominguez et al. demonstrated thatthe molecules, at the concentration of 50 lM, are more potent thatthe DNMTi RG108 but less potent than SGI-1027 [56], a potentnon-nucleoside quinoline-based DNMTi [156]. However, it remainsto determine whether these compounds induce DNA demethyla-tion and to test their anti-cancer potential.

Page 11

192 M. Schnekenburger et al. / Cancer Letters 351 (2014) 182–197

A synthetic derivative of palmitic acid bearing the functionalgroup diynone from the peyssonenyne natural products, called diy-none 8, was reported in vitro to inhibit DNMT1 (30% of inhibition)and activate DNMT3A (a 2.5-fold increase) activities at the concen-tration of 50 lM. Furthermore, this compound induces differentia-tion and apoptosis in U-937 cells starting at the concentration of5 lM. Intriguingly, diynone 8 is toxic to normal human and mousefibroblasts, but not to immortalized human fibroblasts [57]. Thesefindings would require more investigations.

Psammaplins

Psammaplin A (PsA, 21) is a symmetrical brominated tyrosinederived metabolite containing a disulfide linkage formed fromthe condensation of modified tyrosine and cysteine units. Psawas isolated for the first time in 1987 from an unidentified marinesponge [4], probably Thorectopsamma xana [133], from the pacificocean around Guam island and from the sponge Psammaplinaplysil-la sp. from pacific ocean around Tonga [130]. This metabolite wasfurther found in other sponges collected in various places in pacificocean including Aplysinella rhax from Fiji island waters [122],Pseudoceratina purpurea collected from around Papua New Guineaand Madagascar [124] and from a two-sponge association, Poecilla-stra sp. and Jaspis sp. collected from Korean waters [77]. A dimer ofPsA (21), bisaprasin, has also been isolated from T. xana [133] andP. purpurea [124] and many other psammaplin family membershave been isolated of marine sponge of the Verongida order richof such metabolites [122,124,130].

PsA (21) was reported to display broad-spectrum anti-canceractivities. This drug inhibits cell growth and possesses cytotoxicproperties in the low micromolar range against multiple humancell lines from lung, ovarian, skin, colon endometrial and breastcancer subtypes, P-glycoprotein-over-expressing multidrug resis-tant cell lines, and murine and human leukemia cell models[2,55,76,83,107,146]. Notably, PsA (21) radiosensitizes lung cancerA-549 and glioblastoma U-373MG cell lines probably due to aninhibition of DNA repair [85]. The underlying mechanism of PsA(21) anti-cancer properties may rely on its potency to modulatevarious human enzymes that regulate DNA replication, transcrip-tion, differentiation, proliferation, apoptosis, tumor invasion, andangiogenesis. For instance PsA (21) inhibits topoisomerase II [83],farnesyl protein transferase [145], leucine aminopeptidase [145],and polymerase a-primase [76] and activates peroxisome prolifer-ator-activated receptor-c (PPARc) [107]. However, the in vitroinhibitory concentration against these targets is in the micromolarrange. In contrast, it was reported that PsA (21), psammaplin G(22) and bisaprasin inhibit both HDAC and DNMT1 activitiesin vitro in the low nanomolar range, whereas psammaplins B(23), D-F (24–26) and H-J (27–29) were inhibiting only total HDACactivity [124]. Although the in vitro DNMT inhibitory activity of PsA(21) was later observed by another group [60], no DNMT1 inhibi-tory activity was reported in other studies [10,55]. Accordingly,no DNA demethylation has been observed in HCT-116 cells treatedfor 14 days with 1 lM PsA (21) [60]. These discrepancies might berelated to variations in product purity, assay procedure or cellmodels.

Further investigations of the HDAC inhibitory potential of thisclass of compounds point out that psammaplins A (21), B (23), E(25), and F (26) induce histone H3 acetylation at a concentrationof 0.2 lM without affecting a-tubulin acetylation suggesting theyare class I HDACi [84]. Conversely, at the same concentration PsA(21) increases p21 expression associated to cell cycle arrest [2].

Several chemistries starting for instance from tyrosine or phen-ylpyruvic acid derivatives or from aromatic aldehydes (to generatearylpyruvic acids) have been proposed to provide sufficientamounts for further investigations [11,60,69,115] and generated

many analogues allowing deep SAR against HDAC [9,12,55,67,113,120].

These studies point out that PsA (21) acts as a prodrug that sim-ilarly to FK288 need to be reduced in its corresponding monomericthiol form being the active molecule responsible of chelating thezinc ion in the active site of HDACs for their inhibition[10,55,84]. Furthermore, by replacing the a-(hydroxyimino)acylunits by a fluorescent 4-coumarinacetyl moiety, Hentschel et al.demonstrated that the disulfide is cleaved to the thiol probablybefore entering the nucleus [67].

UVI5008 (30) is probably one of the most intriguing PsA ana-logues as in addition to have increased class I HDAC and DNMT3Ainhibitory potential this compound in which a 5-bromoindole unitin 4a replaces the o-bromophenol gained further inhibitory activityagainst SIRT1 and 2. This triple epigenetic regulator at a concentra-tion of 5 lM increases histone, p53 and a-tubulin acetylation andpromotes DNA demethylation in RARb and p16CDKN2A promotersand gene reactivation. Conversely, UVI5008 (30) displays stronganti-cancer properties in several cell lines derived from colon,breast, and prostate carcinomas (IC50 values ranging from 0.2 to3.1 lM) as well as in vivo using a dose of 40 mg/kg in HCT116-or MCF-7-xenografted mice and ex vivo using a concentration of5 lM in acute myeloid leukemia (AML) blasts [113].

Santacruzamate A

Santacruzamate A (31) is a cytotoxin isolated from a cyanobac-teria of genus Symploca sp. collected from the Coiba National Parkon the Pacific coast of Panama. Although santacruzamate A sharesseveral structural features in common with the pan-HDACi sube-roylanilide hydroxamic acid, it acts in vitro at picomolar level asa selective HDAC2 inhibitor [119]. This specificity for HDAC2 couldbe of interest for therapeutic purposes since the general lack ofselectivity of common HDACi is believed to be responsible of manyside effects [118].

Notably, santacruzamate A (31) induces cytotoxicity in colonHCT-116 colon cancer (IC50 = 29.4 lM) and cutaneous T-celllymphoma HuT-78 (IC50 = 1.4 lM) cell lines without affecting theviability of human dermal fibroblasts (IC50 > 100 lM) [119].Nevertheless, it remains to determine whether these effects areconnected to HDAC inhibition in cells.

Streptosetin A

Amagata et al. identified a new class III HDAC inhibitor byscreening a library composed of 506 extracts of marine-derivedactinomycetes employing a HDAC-based yeast assay using anURA3 reporter gene embedded in the telomere region of the yeastchromosome, which is activated by inhibitors of the yeast homo-logue Sir2p to human SIRT1 [3].

Based on a bioassay-guided purification and extensive NMRstudy the new compound was designated as streptosetin A (32),a compound purified from Streptomyces violaceusniger found in asediment sample collected in San Francisco Bay (USA). Furtheranalysis revealed that streptosetin A (32) is an in vitro SIRT1 and2 inhibitor at rather high doses (IC50 values of 3.7 and 4.5 mM,respectively) [3]. Nonetheless, the effect of this compound wasnot tested on cells.

SZ-685C

SZ-685C (33) is a marine anthraquinone purified from man-grove endophytic fungus (No. 1403) collected from the South ChinaSea [167]. This compound was reported to decrease proliferation inmultiple cancer cell models including glioma, hepatoma, breastand prostate cancer cell lines (IC50 values ranged from 3.0 to9.6 lM). Furthermore, this drug induces apoptosis in human breast

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cancer MCF-7 and MDA-MB-435 cells in a dose-dependent mannerfrom 3.75 and 1.5 lM, respectively. Conversely, the intraperitonealinjection of 50 mg/kg SZ-685C (33) inhibits the growth of tumorsformed by MDA-MB-435 cells xenografted in immunodeficientmice. It was suggested that SZ-685C (33) triggers apoptosis bysuppressing Akt/FOXO pathway [167]. Later these data were con-firmed by the investigation of Wang et al., showing that SZ-685C(33) displays pro-apoptotic activity in both radiosensitive and radi-oresistant human nasopharyngeal carcinoma CNE2 cells throughmiR-205-PTEN-Akt pathway [161]. Another study demonstratedthat SZ-685C (33) induces apoptosis in MMQ pituitary tumor cells(IC50 = 13.2 lM) by down-regulating miR-200c, but showed lesstoxicity toward rat pituitary cells (IC50 = 49.1 lM) [24].

Tanikolide dimer

Tanikolide dimer (34) is a dilactone isolated from the marinecyanobacteria Lyngbya majuscula collected near Tanikely Island,Madagascar [66]. Tanikolide dimer was co-purified with tanikolideseco acid using a human SIRT2 bioassay-guided approach. Furtheranalysis revealed that only tanikolide dimer is a potent in vitroSIRT2 inhibitor (IC50 = 176 nM), about 200–300 fold more activethan the well-characterized sirtuin inhibitor sirtinol [66].

Synthetic production of tanikolide dimer (34) and extensivechiral GC–MS approach revealed than the natural tanikolide dimer(34) is (5R, 50R). Comparing naturally occurring tanikolide dimer(34) with other synthetic stereoisomers (5R-5’S, 5S-5S’), Gutierrezet al. observed that chirality does not matter and all three stereo-isomers have similar potential to inhibit SIRT1 and 2 activities,with about 10-fold less potency against SIRT1 than SIRT2 [66]. Sur-prisingly, cytotoxicity assays revealed that tanikolide seco wasmoderately toxic to the human lung H-460 cell line at a concentra-tion of 9.9 lM while tanikolide dimer (34) was inactive at 10 lM.This effect may suggest that tanikolide dimer (34) could be morepromising in neuroprotection [172].

Trichostatins

One of the best-characterized and first natural HDACi identifiedis TSA (35) [89,98]. TSA (35) is a natural hydroxamate originallyisolated from a bacteria present in soil Streptomyces platensis butalso found in other bacterial strains such as Streptomyceshygroscopicus Y-50 and Streptomyces sioyaensis [29,171]. Morerecently, TSA (35) was isolated from a culture of Streptomycesangustmycinicus found associated with a marine sponge collectedfrom a mesopelagic area in Japan waters [70].

TSA (35) was initially reported as a potent inducer of differenti-ation in murine erythroleukemia Friend and RV133 cell lines. Themaximum induction was obtained at 15 nM and 0.5 lM, respec-tively [171]. Later is was reported that TSA (35) inhibits cell cyclein G1 and G2 phases due to an increase of the expression of theCDK inhibitor p21 and can promote differentiation and apoptosisin various solid and non-solid tumors [29]. The anti-cancer proper-ties of TSA (35) are believed to be due to increases histone acetyla-tion as it selectively inhibits in vitro and in vivo the non-sirtuinHDAC enzymes [170].

Besides TSA (35), Hosoya et al. have isolated form the culture ofthe marine-derived bacteria S. angustmycinicus, trichostatic acid(36) and three other trichostatin analogues: JBIR-109 (37), JBIR-110 (38), and JBIR-111 (39) [70]. However, these molecules areover 1000-fold weaker HDAC1 inhibitors than TSA in vitro (IC50

values against recombinant HDAC1 of 35–39 are 0.012, 73, 48, 74and 57 lM, respectively); probably because of the lack of hydroxa-mate functionality, which is of major importance for TSA activityagainst HDACs [170].

Critical considerations and future perspectives

Considering the increasing opportunities for epigenetic modu-lators and marine-derived compounds in anti-cancer therapy, thedevelopment of marine-derived epigenetic modulators is of con-siderable interest. Hence, this review has summarized currentknowledge on the anti-cancer potential of naturally occurring mar-ine compounds capable of affecting several epigenetic mecha-nisms, including DNA methylation, acetylation and methylationof histones, and miRNA effects on gene expression. Nevertheless,so far, there is no HAT or HDM inhibitor reported and most ofthe epigenetically active molecules are targeting HDAC activities.More importantly, most HDACi are reported to be pan-non-sir-tuin-HDACi or selective against HDAC1, 2, 3, 8, 11, which are struc-turally related HDACs. This lack of selectivity might not always betotally correct and could simply represent the consequence of alack of characterization of the HDAC inhibitory activity profilingof considered compounds. Nevertheless, such investigations arecritical to find new scaffolds being more selective against HDACisoenzymes, which could represent valuable tools for mechanisticstudies as well as anti-cancer therapy.

Remarkably, there is no marine or marine-derived compound inclinical trial with well-established epigenetic properties. This lackof compound in clinical trials is probably translating the fact that,to date, the number of epigenetic modulators identified from mar-ine ecosystems is rather limited in comparison to molecules formterrestrial ecosystems, which are mainly plant- and micro-organ-ism-derived compounds. One of the reasons seems most likelybecause compounds from marine origins were under investigatedas potential modulators of epigenetic effectors. However, overthe past few years a number of structures have emerged as inter-esting families of molecules displaying promising anti-cancerproperties. Among these agents, one of the most promising is lar-gazole, which presents one of the most interesting HDAC inhibitoryprofile identified yet associated to a highly effective growth-inhibitory potential in a low nanomolar range. Two other familiesof molecules that sound really attractive are azumamides andpsammaplins. Indeed, the structure variations among the membersof these groups are associated to various HDAC inhibitory profilesand cellular outcomes. These findings may provide new criticaldata for structure–activity relationship studies and the develop-ment of better HDACi with improved specificity and anti-cancerspectrum displaying reduced side effects. Furthermore, psammap-lins as potential dual epigenetic inhibitors may represent leadcompounds for further anti-cancer drug development. Similarly,the identification of TSA analogs provides valuable informationabout structures involved in HDAC inhibition. In this context, it isreasonable to imagine that new TSA analogues could be identifiedin the future with an improved therapeutic potential compared toTSA, which displays a significant toxicity against normal cells andside effects.

Furthermore, considering the important pool of new chemicalscaffolds hold by marine ecosystems and the continuously growingcapacity of exploration of the marine world coupled to an increasinginterest for epigenetic mechanisms and targeted therapies, there isno doubt that new compounds targeting either well-established aswell as newly identified epigenetic targets such as TET proteins willbe discovered and potentially turned into new anti-cancer drugs.Nonetheless, the design of more specific and targeted screening pro-cedures would probably favor the discovery of novel and potentmarine pharmacophores against epigenetic effectors.

The classical epigenetic anti-cancer drugs are commonly target-ing the activity of epigenetic writers or erasers. However, targetingepigenetic readers, which translate the histone code and additionalepigenetic marks constituting the chromatin structure into biolog-

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ical functions could represent an attractive alternative of interven-tion by providing a more targeted approach and finer tuning ofchromatin regulation that targeting in an unspecific mannerHDACs for instance. In this context, modulators of specific epige-netic protein–protein interactions would probably provide newmechanistic insights into chromatin regulation and unravel newtherapeutic opportunities. There are only few examples of suchcompounds in the literature including for instance a series of syn-thetic 4-(pyrrolidin-1-yl)piperidine amine analogues that inhibitselectively the binding of lethal 3 malignant brain tumor-like pro-tein (L3MBTL)1 and L3MBTL3, two methyl-lysine reader proteins ofH4K20me1-2 marks [75]; 4-acyl pyrrole derivative and JQ1 thatinhibit bromodomain containing proteins, which are acetyl-lysinemark readers [44,102]. Nonetheless, the number of potent inhibi-tors of the various histone binding modules and proteins bindingmethylated DNA (i.e. methyl CpG binding proteins (MBDs)) israther limited. Accordingly, using the diversity of scaffolds offeredby the marine world combined to more thorough analyses shouldallow the identification of new potent pharmacophores of thesereaders.

Although the results of anti-cancer strategies based on the use ofvarious epigenetic drugs alone or in combinatory treatments soundvery promising, these approaches require a careful control investi-gation of the impact of such interventions for each classes of epige-netic effectors targeted. Indeed, the remodeling of the epigeneticlandscape may engender adverse side effects such as promotingthe expression of proto-oncogenes and potentially reprogram anyhealthy cells including stem cells, which would be more harm thangood. Nevertheless, we are convinced that the development of suchtherapeutic interventions has a large potential to be furtherincluded in the therapeutic arsenal of clinicians.

Conflict of Interest

Authors declare no conflict of interest.

Acknowledgements

MS is supported by a ‘‘Waxweiler grant for cancer preventionresearch’’ from the Action Lions ‘‘Vaincre le Cancer’’. This workwas supported by Télévie Luxembourg, the «Recherche Cancer etSang» foundation and «Recherches Scientifiques Luxembourg»association. The authors thank «Een Häerz fir Kriibskrank Kanner»association and the Action Lions ‘‘Vaincre le Cancer’’ for generoussupport. MD is supported by the NRF by the MEST of Korea forTumor Microenvironment GCRC 2012-0001184 grant.

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