Natural Bioactive Compounds Targeting Histone Deacetylases in Human Cancers: Recent Updates

Cancer is a complex pathology that causes a large number of deaths worldwide. Several risk factors are involved in tumor transformation, including epigenetic factors. These factors are a set of changes that do not affect the DNA sequence, while modifying the gene’s expression. Histone modification is an essential mark in maintaining cellular memory and, therefore, loss of this mark can lead to tumor transformation. As these epigenetic changes are reversible, the use of molecules that can restore the functions of the enzymes responsible for the changes is therapeutically necessary. Natural molecules, mainly those isolated from medicinal plants, have demonstrated significant inhibitory properties against enzymes related to histone modifications, particularly histone deacetylases (HDACs). Flavonoids, terpenoids, phenolic acids, and alkaloids exert significant inhibitory effects against HDAC and exhibit promising epi-drug properties. This suggests that epi-drugs against HDAC could prevent and treat various human cancers. Accordingly, the present study aimed to evaluate the pharmacodynamic action of different natural compounds extracted from medicinal plants against the enzymatic activity of HDAC.


Introduction
Cancer is a group of pathologies with severe consequences for human health. It is induced by numerous risk factors, such as genetic predisposition, hormonal disorders, oxidative stress, epigenetic instability, microbial infections, and many others [1]. Recently, close correlations between loss of cellular memory, epigenetic instability, and the onset of certain cancers have been demonstrated [2]. In this respect, epigenetic modifications are biochemical marks deposited on DNA and histones, and they participate in the regulation of gene expression at the transcriptional level. These modifications have several roles, including maintaining cell memory during mitotic division; differentiated cells must retain their cellular memory to keep their identity. However, cells can lose their memory and become cancerous due to epigenetic instability. HDACs play a key role in maintaining repressive gene activity. The repressive activity of HDAC leads to the ectopic repression of various genes, which can affect cellular memory. In addition, normal cells can become cancerous after transformation.
The United States Food and Drug Administration (FDA) has approved four HDAC inhibitors as anti-cancer drugs: Vorinostat, Romidepsin, Belinostat, and Panobinostat. In this respect, Vorinostat (Zolinza ® ) was the first histone HDAC inhibitor approved by the FDA in 2006 to treat advanced and refractory cutaneous T-cell lymphoma (CTCL) [3]. Vorinostat binds to the active sites of histone deacetylases and acts as a chelator of zinc ions at the bottom of the HDAC's catalytic domain. It causes chromatin decondensation by inhibiting the suppression of acetyl moieties from the ε-amino groups of the lysine residues of histones and transcription factors [4,5]. It, additionally, has a 30% response rate, as revealed by clinical studies [6]. In November 2009, Romidepsin (Istodax ® ), a second HDAC inhibitor, was approved by the FDA for CTCL, with an overall response rate of 34%, and it was later approved for peripheral T-cell lymphoma (PTCL) [6]. According to published research, Vorinostat inhibits the deacetylation of key autophagic markers and interferes with autophagic cell death, which can be an associated mechanism, depending on the involvement of apoptosis [7]. In July 2014, the FDA approved Belinostat (Beleodaq ® ) as a third HDAC inhibitor, to treat patients with relapsed or refractory PTCL, and which has an overall response rate of 25.8% [8]. Another HDAC inhibitor is Panobinostat (Farydak ® ), which was approved in February 2015, to treat multiple myeloma [9]. Furthermore, other HDAC inhibitors, Valproic acid and Entinostat, are in phase III of a clinical trial for use against solid tumors and hormone-receptor-positive advanced breast cancer [10].
Recent investigations revealed that inhibition of HDAC-like enzymes could prevent and treat different types of cancer at the molecular level, by restoring cellular memory. Within this context, specific secondary metabolites extracted from herbs, including terpenoids, phenolic acids, flavonoids, and alkaloids, have shown inhibitory effects against HDAC [11][12][13][14]. These compounds can act specifically, either against the action of HDAC, or against the signaling pathways induced by HDAC [15,16]. Based on the preceding discussion, this review focuses on the close relationship between epigenetic modifications, particularly HDAC enzymes, and tumor transformation. Thus, the pharmacodynamic action of different natural molecules extracted from medicinal plants against the enzymatic activity of HDAC is discussed, to suggest an innovative therapy implementing the maintenance of cellular memory using anti-HDAC epi-drugs.

Epigenetic Regulation and Cancer
The involvement of environmental, lifestyle, and hereditary factors is the origin of the complexity of cancer etiology. Cancer is most commonly considered a genetic pathology induced by gene expression alteration, following repetitive genetic aberrations. Currently, it is known that the disruption of gene expression causing cell transformation is controlled by epigenetics. Thus, the abnormality of epigenetic regulation has become a model to explain carcinogenesis and cancer development. Research findings suggest that genetic and epigenetic mechanisms are not separate events in cancer, but interact and benefit from each other during tumorigenesis [17,18]. Epigenetic changes are labeled as 'first hits' for tumorigeneses. They are the early events responsible for the loss of tissular homeostasis and induce genetic instability; thus, causing changes in the expression profile of tumorsuppressor genes. Furthermore, it is evident that several tumor suppressor genes are rarely genetically mutated but epigenetically silenced [19].
Epigenetic events include histone modifications (phosphorylation, acetylation, methylation, SUMOylation, and ubiquitylation), DNA methylation, and deregulation of noncoding RNAs and their interactions with proteins or nucleic acids [20]. The dynamic regulation of histone marks, DNA, and chromatin structure is dynamically performed by four types of epigenetic regulators, including (i) writers, (ii) erasers, (iii) readers, and (iv) remodelers [21]. Deregulation and mutations in the genes encoding these epigenetic regulators have been described in various cancers [22].

Cancer and DNA Methylation
DNA methylation consisting of a covalent transfer is the most commonly studied epigenetic modification by DNA methyltransferases (DNMTs) of methyl groups at the fifth carbon of cytosine (5-mC) within 5 -CpG-3 di-nucleotides [23,24]. In mammals, DNMT1, DNMT3a, and DNMT3b are the three main classes of DNMT enzymes. In this regard, abnormal DNA methylation patterns are due to DNMT overexpression or aberrant recruitment. Cancer cells exhibit abnormal DNA methylation patterns, marked by global hypomethylation associated with the promoter and focal hypermethylation of specific genes [25,26]. In addition, aberrant hypomethylation induces the expression of numerous genes, including oncogenes [27], whereas hypermethylation inhibits specific tumor suppressor genes (TSGs) [28]. On the other hand, hypomethylation of oncogenesis is often revealed in cancers such as SLC34A2 in papillary thyroid carcinoma, LY6K in glioblastoma, and RBBP6 in colorectal cancer, among others [29][30][31].
Moreover, hypermethylation is easily observed at precancerous stages in benign tumors and tumor-predisposing inflammatory lesions [32,33]. In this respect, the retinoblastoma gene's hypermethylation of the CpG island promoter is released in retinoblastoma [34]. Several research studies have shown promoter hypermethylation and silencing of other TSGs in renal cancer; VHL (von Hippel-Lindau) [35] in bladder cancer; the cell cycle regulator CDKN2 A/p16 [36]; and in colon cancer, the mismatch repair gene hMLH1 [37]. Additionally, abundant hypermethylated TSGs include RASSF10 and SIX3 in kidney cancer and glioblastoma, respectively [38,39]; PTEN, and CDKN2A in melanoma [40,41]; and CDKN2A, TIMPS, and DAPK in prostate cancer [42]. In contrast, significant methylation of EN1 and SCTR was observed in the prostate, colorectal, and salivary gland adenoid cystic carcinoma [43]. The gene APC is hypermethylated in pancreatic cancer [44]. Indeed, hMLH1 still carries a genetic mutation and hypermethylation of an allele in the colon cancer cell line HCT116, which induces the inactivation of the main tumor suppressors [45]. Moreover, pax6, p16, and p15 are generally aberrantly methylated in bladder cancer [46].

Oncohistones and Histone Changes
Histone mutations play an important role in cancer epigenetics, where recurrent mutations targeting histone genes have been described in several types of cancer. Genes coding for histones are mostly mutated in cancer and are commonly named 'oncohistones' [47]. Mutations affecting all canonical histone classes and non-canonical histones have been recorded in different tumors [47]. H2A and H2B mutations occur in carcinosarcomas, while H1 mutations were detected in diffuse large B cell lymphomas [47,48]. Similarly, mutations in H3 and its non-canonical counterpart H3.3 were seen in children's tumors [49,50], whereas mutations in osteosarcoma and giant cell tumor of bone harbor H3.3 G34W mutations [51,52].

Histone Methylation and Cancer
Histone methylation affects the affinity of transcription factors, leading to activation or restriction of transcription. Irregularities in the methylation of different lysine residues can modify gene expression [57]. In general, H3K4, H3K36, and H3K79 are the most important sites where their methylation causes gene transcription, while methylation of H3K27, H4K20, and H3K9 is related to transcription silencing [58]. In addition, trimethylations of H3K79, H3K27, and H3K9 induce repression, while mono-methylations of H4K20, H3K27, H3K9, H2BK5, and H3K79 induce gene expression.

Histone Acetylation/Deacetylation and Cancer
Histone acetylation and deacetylation are correlated with the active and open chromatin conformation and inactive and condensed chromatin form, respectively. In this respect, the HDAC and HAT enzymes control the regulation of histone acetylation, which is very dynamic [67]. The acetyl-CoA acetyl group was added, by HAT, to the histone at the lysine position, which neutralizes the positive lysine charge disrupting the electrostatic interaction between DNA and histones. The chromatin structure was attenuated; thus, affecting the gene assembly and changing the transcription operation [68].
An imbalance of histone acetylation was detected in lung cancers, Rubinstein-Taybi syndrome, AML, and glioblastomas [69]. Many members of HATs mutate differently in tumors [70][71][72] and participate in different stages of its development, including B-cell non-Hodgkin lymphoma and leukemia [73], and have also been described in solid cancers [74,75]. Furthermore, chromosomal translocations implying HATs and their fusion proteins were involved in the onset and development of acute leukemia [76]. Moreover, any disturbance in the expression of distinct HDAC isoforms induces different cancers. Indeed, dysregulation of HDAC proteins causes aberrant deacetylation and inhibition of TSGs. HDACs can also regulate gene transcription via deacetylating the DNMT, HAT, and HDAC proteins responsible for epigenetic events [77].

Phosphorylation, Ubiquitination, SUMOylation, and Cancer
Abnormalities in kinase activity lead to a variety of cancers [78,79]. The decreased activity of E3 ubiquitin ligase caused by specific mutations can be the origin of various tumors, such as breast cancer and renal cell carcinoma. Moreover, cervical cancer is caused by an increase in the ubiquitination effect, while glioblastoma and colorectal cancer are induced by the total elimination of ubiquitination [80]. SUMOylation is a process required for all cells, and not as a tumor promoter and suppressor [81].

Epigenetic Regulation by miRNAs and Cancer
Epigenetic linked noncoding RNAs (ncRNAs) comprise small interfering RNA (siRNAs), long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and Piwi-interacting RNA (piRNAs). In this regard, ncRNAs are involved in complex double-negative feedback loops, where miRNA inhibition of an epigenetic regulator is performed at the epigenetic level by the same regulator. In contrast, the epigenetic modifier enzymes involved in epigenetic modulation can be targeted by miRNAs, establishing a trilateral regulatory 'epi-miR-epi' feedback circuit. The intricate interaction between the epigenetic architecture and miRNAs is important to surveilling gene expression profiles in cancer [82]. They have been classified as tumor suppressors, oncogenic, or context-dependent miRNAs [83]. Furthermore, miRNAs regulate the expression of epigenetic regulatory enzymes, such as HMT, DNMT, and HAT. Moreover, aberrant miRNA profiling indicative of altered regulatory factors, including cell migration and proliferation, has been reported in numerous tumors, with the most manifest being the decreased ncRNA expression levels in cancer cells compared to normal tissues [84].

The Role of HDAC in Cancer
In many human cancers, dysregulation of epigenetic enzymes results from mutations, abnormal expression, and/or disproportionate recruitment to certain loci. Histone acetylation modulates the chromatin structure, which is a key factor regulating gene expression. Histone acetylation is due to the well-balanced activities of HATs and HDACs. Indeed, HDACs remove the acetyl moiety from lysine residues, resulting in positively charged histones. This increases the ionic interactions with DNA that provoke a compacted chromatin structure, which represses gene expression by making it difficult to access the transcription machinery. Moreover, in the absence of a signal, HDACs can form a corepressor complex with the nuclear receptor and interact directly with transcription factors (Figure 1) [98].

Epigenetic Regulation by miRNAs and Cancer
Epigenetic linked noncoding RNAs (ncRNAs) comprise small interfering RNA (siR-NAs), long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and Piwi-interacting RNA (piRNAs). In this regard, ncRNAs are involved in complex double-negative feedback loops, where miRNA inhibition of an epigenetic regulator is performed at the epigenetic level by the same regulator. In contrast, the epigenetic modifier enzymes involved in epigenetic modulation can be targeted by miRNAs, establishing a trilateral regulatory 'epi-miR-epi' feedback circuit. The intricate interaction between the epigenetic architecture and miRNAs is important to surveilling gene expression profiles in cancer [82]. They have been classified as tumor suppressors, oncogenic, or context-dependent miRNAs [83]. Furthermore, miRNAs regulate the expression of epigenetic regulatory enzymes, such as HMT, DNMT, and HAT. Moreover, aberrant miRNA profiling indicative of altered regulatory factors, including cell migration and proliferation, has been reported in numerous tumors, with the most manifest being the decreased ncRNA expression levels in cancer cells compared to normal tissues [84].

The Role of HDAC in Cancer
In many human cancers, dysregulation of epigenetic enzymes results from mutations, abnormal expression, and/or disproportionate recruitment to certain loci. Histone acetylation modulates the chromatin structure, which is a key factor regulating gene expression. Histone acetylation is due to the well-balanced activities of HATs and HDACs. Indeed, HDACs remove the acetyl moiety from lysine residues, resulting in positively charged histones. This increases the ionic interactions with DNA that provoke a compacted chromatin structure, which represses gene expression by making it difficult to access the transcription machinery. Moreover, in the absence of a signal, HDACs can form a corepressor complex with the nuclear receptor and interact directly with transcription factors (Figure 1) [98]. So far, 18 mammalian HDACs have been described and assembled into four families according to their homology with yeast HDACs [99,100]. Homologous to yeast Rpd3, class So far, 18 mammalian HDACs have been described and assembled into four families according to their homology with yeast HDACs [99,100]. Homologous to yeast Rpd3, Class I HDACs (1, 2, 3, and 8) are localized in the nucleus of human cells. Class II HDACs (4, 5, 6, 7, 9, and 10) are homologous to yeast Hda1, exhibit a tissue-specific expression, and can shift between the nucleus and cytoplasm. Class III HDACs, or sirtuins (SIRT1-7), are homologous to yeast Sir2 and require the NAD+ cofactor for their activity. Finally, Class IV HDACs, including the recently discovered HDAC11, display the characteristics of both Class I HDACs and HDAC II [99,100].
The implications of HDACs in cancer development were first reported in hematological malignancies by inappropriate involvement of HDAC-containing complexes [100,101].
Until now, very rare mutations altering HDAC expression and activity have been recorded in tumors, while deregulation of their activity has been associated with abnormal gene expression and carcinogenesis. Many studies reported that HDAC1 is overexpressed in prostate, colon adenocarcinoma gastric, and breast carcinomas [102][103][104][105], whereas HDAC2 is overexpressed in colorectal [106], cervical [107], and gastric cancers [108]. Overexpression of HDAC1, HDAC2, and HDAC3 is linked to low survival in patients with gastric and ovarian cancers, while HDAC6 was highly expressed in breast cancer specimens [109]. HDAC8 overexpression was reported in neuroblastoma, whereas low HDAC4 levels are reported in gastric cancers [110,111].

HDAC in Different Cancer Stages
During cancer development, many factors, including inhibition of apoptosis and differentiation, and the promotion of proliferation, angiogenesis, and metastasis are epigenetically regulated. Here, we tried to associate the deregulation of HDACs with different steps of tumor development.

Cell Cycle Progression and Apoptosis
Several HDACs (from 1 to 6) are involved in tumor development, and their loss promotes cell proliferation dysregulation [113,116,[127][128][129]. HDAC1 reduces suppressors of the cell cycle mutually with Rb and by altering E2F1 activity [130]. The inhibition of HDAC 1-2 induces cell cycle arrest [131]. HDAC1 is also involved in G 1 /S and G 2 /M transitions. Another study also showed that HDAC1 knock-down contributes to G 2 /M phase arrest [132]. Similarly, both HDAC3 and HDAC10 modulate the G 2 /M transition [133,134]. High levels of Sp1 due to HDAC1/2/6 activities promote the division of cancer cells and G 2 /M progression [135]. Knockdown of HDAC3 induced gathering of cells at the G 2 /M phase [103], whereas in osteosarcoma cells, this effect causes siRNA-mediated HDAC1 depletion [129].
Cell cycle interruption at the G 2 /M stage in renal cancer, following the inhibition of HDAC6 and HDAC3, has been ascribed to proteasomal alteration of Aurora B and A [136]. Furthermore, SIRT1 can suppress the cell cycle through the blockage of p53-dependent pathways [137]. HDAC11 negatively affects E2F7, E2F8, and cell cycle suppressors, leading to survival of tumor cells [138]. Moreover, HDACs act as apoptosis regulators, as the interruption of this process is a critical factor for tumor progression and, therefore, is considered a hallmark of tumor progression. HDACs contribute to the extrinsic, as well as intrinsic, apoptotic pathways. Regarding the extrinsic apoptotic pathways, HDACs can obstruct TRAIL or TGF-b-mediated pathways, while pro-and antiapoptotic factors are altered in the intrinsic pathway [139].

Differentiation
During differentiation, establishing a specific gene expression profile is harmonized by epigenetic modifications, e.g., histone acetylation. In this context, HDAC3 was recruited by RARPML [140], while HDAC4 interacted with RAR-PLZF [141] to repress differentiation-specific transcription. A close mechanism of retinoic acid signaling limi-Molecules 2022, 27, 2568 7 of 43 tation in hematopoietic cells was recorded in AML1-ETO fusion proteins, which bind to HDAC1, 2, and 3 [142,143]. HDAC8 is a key regulator of cancer cell differentiation [144], and HDAC8 overexpression is associated with neuroblastoma progression.

DNA Damage Response
HDACs contribute to DNA damage repair (DDR) responses via their key role in remodeling chromatin and regulating the acetylation patterns of proteins associated with DNA [145]. The inhibition of HDAC blocks double-strand break (DSB) repair and radiosensitizes cancerous cells. In this line, HDAC2 and HDAC1 bind to DNA damage regions, to deacetylate histones at H3K56 and H4K16 and promote non-homologous end-joining pathways, which accelerate DSB repair [146]. Moreover, HDAC3 is involved in nucleotide excision repair (NER) [147], whereas HDAC9 and HDAC10 contribute to homologous recombination [148,149]. HDAC6, in association with DNA mismatch repair protein (MSH2), acts as an MSH2 inhibitor through deacetylation and ubiquitination [150]. In addition, Sirtuins interact with numerous proteins regulating several DDR pathways [151]. In tumor cells, SIRT1 restraints p53 acetylation, contributing to cell survival [148,152]. SIRT6 phosphorylation is directly engaged in DNA damage sites to promote DSB repair [153,154]. In leukemia-initiating cells, the inhibition of SIRT6 or HDAC8 engenders a DNA repair deficit in homologous recombination and the NHEJ pathway [155].

Metastasis
The capacity to disperse and metastasize represents the deadliest signature of tumor cells. Numerous works have evidenced that HDACs regulate metastasis in various cancers. An important player in metastasis is the transition from adherent epithelial cells to motile mesenchymal cells capable of leaving the primary tumor site. In embryonic development, EMT is a crucial path for cell migration during gastrulation [156]. In colorectal cancer, HDAC3 was engaged to Runx 2 promoter and hampered metastasis [157]. Research findings showed that HDAC7 enhances EphA2 expression by downregulating miR-4465 expression, positively affecting tumor proliferation, spread, and invasion in nasopharyngeal cancer [158]. Similarly, HDAC11 leads to the overexpression of RRM2, a gene involved in promigratory and metastatic phenotypes [138]. In prostate cancer, ZEB1 and SIRT1 together bind to CDH1, promoting metastasis [159], while SIRT1 elicited EMT through Fra-1 over-expression in colorectal cancer [160]. Nonetheless, SIRT1 seems to limit metastasis, not only via deacetylation of Smad4, but also via repression of TGF-b-triggered signaling, whereas SIRT2 deacetylated Slug and stabilized its protein to increase EMT [161]. Many metastasis repressors, such as RECK, Kangai 1, RhoB, and TIMP-1, increased their level in response to HDAC inhibitors, indicating a potential role of HDACs in the promotion of metastasis [162]. In addition, HDAC1 impaired the expression of RECK and RhoB in cancer cells [163]. EMT is promoted by transcription factors of the SNAIL/SLUG family, mediating the silencing of E-Cadherin expression by engaging the SIN3/HDAC1/HDAC2 corepressor complex and upregulating the matrix metalloproteinase family proteins. Furthermore, SNAIL1, by interacting with HDAC1-2, inhibits the E-Cadherin promoter, since the addition of trichostatin A (TSA) obstructs the effect of SNAIL1 in SNAIL1-expressing epithelial cells [164]. The pivotal role of SNAIL1 in association with HDAC1/HDAC2 and resulting in E-Cadherin repression in cancers has been documented [165].

Angiogenesis
Angiogenesis involves the creation and addition of new blood vessels and is pivotal for the development of tumors [166,167]. The first steps of angiogenesis are elicited by hypoxia or a hypoxic microenvironment, while its advancement is mainly controlled by hypoxia-inducible factor 1a (HIF-1a). Overall, HDACs monitor the balance between proand anti-angiogenic proteins. In this context, HDAC inhibition exerts anti-angiogenic activity via inhibition of pro-angiogenic gene expression. Under hypoxia conditions, Class I HDACs, mRNA, and protein were overexpressed in vitro in primary and malignant cells [168]. HDAC1 deacetylates HIF-1a, contributing to preventing HIF-1a loss. On the other hand, dysregulated levels of HDAC1 lead to high levels of HIF-1a and VEGF in tumors, which in turn enhances angiogenesis [169]. HDAC4, 6, 10, and SIRTs display similar pathways [139], whereas HDAC4, 5, and 6, acting as mediators of HIF-1 activity, require cofactors (HSP90 and p300) [170]. On the contrary, it was shown that SIRT1 deacetylated HIF-1a, which reduces the interaction of HIF-1a with p300, which reduces HIF-1a activity. In endothelial cells, HDAC5 reduced the expression of pro-angiogenic genes (FGF2 and Slit2) [171]. Additionally, HDAC5 represses cysteine-rich angiogenic inducer 61 (CYR-61), a well-known antifibrotic and pro-angiogenic mediator, inhibiting angiogenesis [172]. HDAC6 enhances angiogenesis via deacetylation of cortactin, an actinremodeling protein [173].

Autophagy
Autophagy is a process that suppresses damaged subcellular fractions, helping to intercept the transformation of normal cells to cancerous ones [174,175]. Published data showed that Class I HDACs mediate autophagic flux in mice [50], whereas elimination of HDAC1 and HDAC2 impedes autophagic flux [176]. On the other hand, HDAC4 and HDAC5 influence autophagic flux by acting as positive regulators of tumor cell development. HDAC6 promotes autophagy, based on its connection with microtubule proteins [177,178]. In this regard, autophagy might be actively enhanced and play a compensatory role for HDAC6 under ubiquitin-proteasome system damage [120,148]. At the same time, HDAC6 shows a significant role in ubiquitin-selective quality control autophagy, instead of starvationinduced autophagy [163]. Similarly, Parkin-mediated mitochondrial ubiquitination could engage the autophagic actors, i.e., HDAC6 and p62 [179]. HDAC10 knock-down induces autophagosome/lysosome fusion blockade and restriction of autophagic flux, which sensitizes cells to chemotherapy [180]. HDAC10 also deacetylates HSP70 protein members associated with autophagy-mediated cell longevity [180]. In contrast, SIRT1 displays a dual role in autophagy [181], where it is necessary to trigger starvation-induced autophagy [127,182]. Moreover, SIRT1 deacetylates forkhead box O3 (FOXO3), leading to proteasomal degradation and, thus, contributing to the overexpression of numerous autophagic genes.
In embryonic stem cells (ESCs), SIRT1 affects the PI3K/Beclin 1 and mTOR pathways, affecting oxidative stress-induced autophagy [183]. SIRT2 detaches from FOXO1 under stress conditions, which promotes hyperacetylated FOXO1, promoting the autophagic process [184]. In contrast, deacetylation of lactate dehydrogenase B (LDHB) by SIRT5 intensifies its effect. Protons (H + ) generated by LDHB promote autophagy in tumor cells [181,185]. Furthermore, SIRT5 is involved in ammonia-induced autophagy, via glutamine metabolism remodeling [184]. SIRT6 promotes autophagy by hampering the transcriptional repressor Nkx3.2, resulting in the expression of GATA5 [186].  Table 1), a flavonoid found in green tea, exhibits potent anticancer properties, by targeting epigenetic mechanisms, especially HDAC pathways. Published research showed that EGCG reduced the expression levels of HDAC2 and HDAC3, by 50 µM to 150 µM for 48 and 72 h [187], as well as downregulating the HDAC1 protein level by approximately 50% at 100 µM [188] in human colon cancer cell lines. Treatments with EGCG (40 µmol/L) and a pro-drug EGCG (pEGCG) (20 µmol/L) induced a significant suppression in the proliferation of both MCF-7 and MDA6MB 231 breast cancer cells via epigenetic mechanisms, as it inhibited the activities of histone acetyltransferase (HAT) [189]. This compound can remodel the chromatin structures of the hTERT promoter by downregulating acetyl-histone H3 and H4, as well as acetyl-H3K9 expression levels, accompanied by chromatin alterations, leading to the binding of different hTERT repressors, including E2F-1 and MAD1 to the hTERT regulatory region [189]. In skin cancer A431 cells, EGCG (25 µM) has been reported to induce an increase in acetylated lysine 14 and 9 (on histone H3) and acetylated lysine 12, 5, and 16 on histone H4 (H3-Lys and H4-Lys) for six days [190]. Recently, it was found that EGCG (200 µM) can increase H3K9/14ac and H3ac histone acetylation up to 8-fold compared to the reference, accompanied by concomitant hypermethylation of the active H3K4me3 (almost 2.5-fold) and the repressive H3K9me3 chromatin proteins (up to 160%), correlating with downregulation of heterochromatin binding factors such as HP1α and HP1γ [191].

Natural Bioactive Compounds Targeting HDAC in Human Cancers
of HDAC2 and HDAC3, by 50 μM to 150 μM for 48 and 72 h [187], as well as downregulating the HDAC1 protein level by approximately 50% at 100 μM [188] in human colon cancer cell lines. Treatments with EGCG (40 μmol/L) and a pro-drug EGCG (pEGCG) (20 μmol/L) induced a significant suppression in the proliferation of both MCF-7 and MDA6MB 231 breast cancer cells via epigenetic mechanisms, as it inhibited the activities of histone acetyltransferase (HAT) [189]. This compound can remodel the chromatin structures of the hTERT promoter by downregulating acetyl-histone H3 and H4, as well as acetyl-H3K9 expression levels, accompanied by chromatin alterations, leading to the binding of different hTERT repressors, including E2F-1 and MAD1 to the hTERT regulatory region [189]. In skin cancer A431 cells, EGCG (25 μM) has been reported to induce an increase in acetylated lysine 14 and 9 (on histone H3) and acetylated lysine 12, 5, and 16 on histone H4 (H3-Lys and H4-Lys) for six days [190]. Recently, it was found that EGCG (200 μM) can increase H3K9/14ac and H3ac histone acetylation up to 8-fold compared to the reference, accompanied by concomitant hypermethylation of the active H3K4me3 (almost 2.5-fold) and the repressive H3K9me3 chromatin proteins (up to 160%), correlating with downregulation of heterochromatin binding factors such as HP1α and HP1γ [191].   Table 1) is a yellow crystalline solid flavone with several natural sources, including medicinal plants. In human prostate cancer PC-3 cells, apigenin (20-40 µM) acts as a potent HDAC inhibitor, which induces noticeable suppression of the HDAC protein activity (41% and 62%), especially HDAC1 and HDAC3 [192]. It additionally promoted global acetylation of both histone H3 (7.4-and 8.2-fold) and H4 (1.2-and 2.6-fold), as well as specifically centered the hyperacetylation of histone H3 on the p21/waf1 promoter [192]. In this context, Tseng et al. [193] demonstrated that apigenin treatment for 48 h could inhibit the HDAC activity in breast cancer cells. Moreover, molecular analysis revealed that apigenin increases the expression levels of acetylated H3, particularly in the p21 WAF1/CIP1 promoter area mediated by the upregulation of p21 WAF1/CIP1 transcription [193].

Galangin
Galangin ( Figure 2, Table 1) is a natural flavonoid found in honey and in lesser galangal (Alpinia officinarum Hance.). This natural compound exhibits a promising effect against HDAC activity. Treatment with galangin for 24 h increased endogenous HDAC1-mediated deacetylation, independent of DNA methylation status, and subsequently decreased histone H3 acetylation in the BACE1 promoter regions in human neuroblastoma SH-SY5Y cells [194].

Genistein
Genistein ( Figure 2, Table 1) is a naturally occurring isoflavone, first isolated from Genista tinctoria. This compound is known for its promising chemotherapeutic effect against several types of cancer. Genistein (5-20 µmol/L) showed, in vitro, a weak inhibitory capacity of HDAC catalytic activity (13-17%) in human esophageal squamous carcinoma cells [195]. HAT and HDAC analysis demonstrated that genistein decreases HDAC, but increases HAT activity. ChIP analysis with various antibodies showed enrichment of acetylated histones H3, H4, and H3 di-and tri-methylated lysine 4 in prostate carcinoma cell lines (LNCaP, PC3) [196]. On the other hand, using mouse models, Li and coworkers [197] reported that a diet containing 250 mg/kg genistein reduced HDAC1 expression in vivo at protein and mRNA levels. Moreover, genistein also suppressed the phosphorylation of serine ten and methylation of lysine nine at the promoter regions of various genes, such as Wnt5a, Sfrp5, and Sfrp2 [198].

Quercetin
Quercetin ( Figure 2) can be found in medicinal plants and numerous food products. This compound possesses different biological activities, including anticancer, via various mechanisms of action. Quercetin alone, or combined with other compounds, exhibited effective epigenetic modifications. At a concentration of 100 µM, it increased the histone H3 acetylation mediated by the upregulation of FasL expression, and activated the HAT and inhibited the HDAC activities [202]. In addition, quercetin (25 and 50 µM) modulated the expression of numerous chromatin modulators after 48 h of treatment and decreased HDAC and HMT activities, including the activity of HMT-H3K9 [203]. Listed in Table 1 are the natural flavonoids targeting HDAC in human cancers. In the post-AOM phase, there was a decrease in H3Ac at the promoter of Wnt5a, Sfrp5, and Sfrp2. Repressed histone H3 lysine 9 methylation and serine 10 phosphorylation at the promoters of Sfrp2, Sfrp5, and Wnt5a in the post-AOM period.
In the post-AOM period, the nuclear level of HDAC3 protein was increased. After AOM induction, H3Ac was reduced in the same region of the Sfrp5 promoter. [198] Genistein (

Alkaloids
Alkaloids such as berberine, nicotinamide, isofistularin-3, psammaplin, and TSA (Table 2, Figure 3) exhibit promising anticancer effects via the control of epigenetic changes and various other mechanisms. The HDAC signaling pathways represent some of the main targets of these bioactive compounds. In this context, berberine (120 µM) exhibits remarkable anticancer effects via targeting epigenetic chromatin modification, which affects the enzyme implicated in histone acetylation and methylation in acute myeloid leukemia (AML) cell lines [224]. These events revealed the role of berberine as a master epigenetic regulator responsible for controlling downstream bioactivities, including cell cycle arrest, apoptosis, and the underlying mechanism of metabolic disorders and immune response.  Reduced the activity of HDAC, either alone or in conjunction with GEN. [197] TSA (Purchased) SKOV3 (Ovarian cells) Western blot analysis Histone immunoblots assay TSA + decitabine reduced the DNMT and HDAC activities. TSA + decitabine increased acetylation of histone H3 and H4. TSA + decitabine inhibited the expression of lysine-specific demethylase-1. TSA + decitabine increased the transcription activity marker dimethylated-H3K4, while suppressing dimethylated-H3K9. [231] The findings by Florean and colleagues [225] revealed that isofistularin-3, a brominated alkaloid derivative isolated from Aplysina aerophoba, can inhibit DNMT1 in vitro (IC 50 = 13.5 ± 5.4 µM), through binding to the DNA-interacting pocket of the enzyme. In addition, Iso-3, at a dose of 25 µM, induced modification of the AHR promoter methylation and upregulated the AHR expression in RAJI cells. These effects are probably associated with generating an altered chromatin state via histone deacetylase activity (HDAC). These results were validated in vivo in a zebrafish xenograft model. Notably, researchers provided evidence that Iso-3 does not elicit acute toxicity in developing zebrafish, shown by the absence of morphological defects and dead embryos, even at the highest dose, after 24 h of treatment. Moreover, using a mice skin model, Tiwari and Gupta [226] found that nicotinamide (NA) treatment (5 mg) increases the expression levels of HDACs, mediated by the downregulation of tumor suppressor miR-203, at a period between 4 and 16 weeks. The combination of this molecule (NA) with 7, 12-dimethylbenz [a] anthracene, a potent carcino-gen compound, exhibited further effects: targeting the HDAC pathway, providing a novel chemoprevention strategy by combining different compounds through the control of epigenetic and microRNA biogenesis modulators in tumor development, in a time-dependent manner [226]. Within this context, the study of Vincent et al. [227] demonstrated that TSA (0.3 µM), a hydroxamic acid first identified in the bacterium Streptomyces platensis, exhibits a broad-spectrum of reversible HDAC inhibitory activity. Chromatin immunoprecipitation and RNA interference assays showed that TSA inhibits the histone deacetylation concomitant with potent MUC4 repression in high-expressing cells [227]. Moreover, treatment of breast MDA-MB-231 cell lines with TSA (100 ng/mL for 12h) combined (25 µM for two days) with genistein showed promising histone changes in the estrogen receptor-α (ERα) promoter and consecutively decreased the HDAC activity [197]. The potential toxicity of this new combination has been reported in normal human mammary epithelial cells (HMECs). It has been shown that the combined treatment of TSA with genistein is potentially safe and can be applied to in vivo experiments.
In a similar fashion, Ahn and coworkers [228] reported the antiproliferative activity (IC 50 = 5µg/mL) of the alkaloid psammaplin A as an HDAC suppressor. They showed that this molecule exhibited remarkable epigenetic regulation and exhibited antiproliferative activity against endometrial cancer cells at the lowest dose of 5 µg/mL, triggering genes related to apoptosis and cell cycle arrest, accompanied by accumulation of acetylated histones and decreased expression levels of HADC [228]. Moreover, using human cancer cell lines, Baud et al. [229] reported the molecular and the enzymatic mechanisms of psammaplin A against its HDAC targets. These researchers showed that psammaplin A (11c) remarkably affects HDAC1 in vitro (IC 50 = 0.9 nM). Regarding the enzymatic specificity of psammaplin A, it has been found that this compound exhibits strong isoform selectivity, which is 360-fold selective for HDAC1 compared to HDAC6, and more than 1000-fold less effective against HDAC8 and HDAC7.

Corosolic Acid
Corosolic acid (Figure 4, Table 3) is a triterpene acid isolated from Lagerstroemia speciose. This bioactive compound is commonly abundant in food sources such as guava, loquat, and olive, and exhibits various pharmacological effects, including anti-proliferative activity. In this respect, Zhang and coworkers [232] studied the role of corosolic acid in the activity and expression of HDACs, in a transgenic adenocarcinoma mouse prostate (TRAMP) model, as well as its involvement in DNA and histone H3 methylation. These researchers found that corosolic acid promotes downregulation of the expression and activity of epigenetic modulatory proteins, demonstrating its ability to prevent prostate carcinogenesis. In addition, corosolic acid (4 µM) significantly increased the expression levels of the acetylated histone H3 lysine 27 (H3K27ac), accompanied by a decrease in histone H3 lysine 27 trimethylation (H3K27Me3) at Nrf2 promoter in TRAMP-C1 cells [232].

Cucurbitacin B
Cucurbitacin B (Figure 4) is a plant-derived triterpene, initially found in the cucurbitaceae plant species. Published research has indicated that this molecule acts as a potent HDAC inhibitor at sub-IC 50 and at IC 50 s in the nanomolar range (IC 50 = 60 nM) for nonsmall cell lung cancer cells (NSCLC H1299 cells). Cucurbitacin B induced suppression of the activity and expression levels of epigenetically modifying marks. It also produced histone changes at the p16 INK4A , p21 CIP1/WAF1 , and hTERT promoters in human NSCLC H1299 cells [233]. An in vivo experiment using a mouse model for lung cancer showed that cucurbitacin B at a concentration of 0.2 mg/kg b.w significantly inhibits the expression levels of different HDAC proteins (1)(2)(3)(4), without visible signs of toxicity in animals. main targets of these bioactive compounds. In this context, berberine (120 μM) exhibits remarkable anticancer effects via targeting epigenetic chromatin modification, which affects the enzyme implicated in histone acetylation and methylation in acute myeloid leukemia (AML) cell lines [224]. These events revealed the role of berberine as a master epigenetic regulator responsible for controlling downstream bioactivities, including cell cycle arrest, apoptosis, and the underlying mechanism of metabolic disorders and immune response.

Ursolic Acid
Ursolic acid (3-beta-3-hydroxy-urs-12-ene-28-oic-acid) ( Figure 4) is a triterpenic acid distributed in different plants and foods, such as ginseng (Panax Ginseng), rosemary (Rosmarinus officinalis), apple peel, pear, cranberry, and plum (Prunus domestica) [234,235]. It has been widely explored for its chemopreventive and chemotherapeutic effects on various cancers. Ursolic acid has been investigated for its action against different epigenetic regulators, including HDAC proteins. Research findings showed that this bioactive compound considerably downregulates the expression of several epigenetic modulatory factors, starting at a dose of 2.5 µM (non-toxic concentrations), including HDAC1, HDAC2, HDAC3, and HDAC8 (Class I), in addition to HDAC6 and HDAC7 (Class II) [232]. Ursolic acid also suppressed induction of HDAC1 and HDAC3 markers in leukocytes, mediated by LPS in mouse epidermal cells [232].      Butyrate is a short-chain fatty acid, mainly generated during the fermentation of carbohydrates by the intestinal microflora [242]. This molecule exerts an efficient epigenetic effect, acting as an HDAC inhibitor (Table 4). In this respect, Saldanha and coworkers [206] reported that butyrate induces important epigenetic modifications at relatively high, but physiologically achievable, concentrations (5 mM), as it affects the global DNA methylation and chromatin structure; thus, inhibiting HDAC1 expression in RKO, HCT-116, and HT-29 colorectal cancer cell lines. Moreover, butyrate decreased the reverse expression of HDAC1 in human esophageal 9706 cancer cells [223]. The combination of butyrate with quercetin showed the reverse effect, triggering remodeled DNA methylation and histone acetylation; thus, inhibiting HDAC activity [223]. This effect was mediated by NF-κB epigenetic signaling cascades. Generally, butyrate, through its epigenetic regulatory effects on HDACs, exhibits functions as a modulator of chromatin structure and may mediate cellular responses via up-regulatory genes responsible for cell cycle arrest, promoting therapeutic outcomes.

Butyric Acid and Eicosapentaenoic Acid
Butyric and eicosapentaenoic acids are fatty acids with several vegetable sources and exhibit important biological properties, including anticancer activity with different mechanisms, such as their effect on epigenetic targets (Table 4). Tiwari and Gupta [226] reported, in vivo, the antitumor activity of butyrate alone (17.62 mg) or combined with other compounds, including nicotinamide (NA) (5 mg) and calcium glucarate (CAG) (5 mg). The results showed that butyric acid exhibits a promising effect on 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumor through increasing HDAC expression and activity, accompanied by upregulation of miR-203 promoter methylation at 4 or 16 weeks [226]. This effect was intensively promoted by co-administration of butyric acid with NA and CAG, targeting epigenetic or biogenetic modulations. On the other hand, treatment with eicosapentaenoic acid (100 µM) promoted the expression of the tumor suppressor gene by downregulating the expression levels of the HDAC1 enzyme, mediated by the activation of peroxisome proliferator-activated receptor (PPARγ) in hepatocarcinoma cells (HCC). Phenethyl isothiocyanate (PEITC) ( Figure 5, Table 5), is a secondary plant metabolite, naturally present as gluconasturtiin in numerous cruciferous vegetables, and has shown remarkable anticancer properties via multiple mechanisms, including as an epigenetic regulator. In one study, PEITC was used at multiple doses (0-20 µM). Long-term PEITC treatment (6 weeks; 2.5 µM) resulted in a 40% reduction in viability compared to cells exposed to the DMSO vehicle alone, but similar effects were not evident in a culture of 72 h, demonstrating that 2.5 µM PEITC is not highly cytotoxic in short-term cultures [244].
Several HDACs, including HDAC1, HDAC3, SAP18 (sin3-associated protein 18), and SAP30 (sin3-associated protein 30), showed preferential attachment to heterochromatin after exposure to PEITC, indicating that PEITC may prevent HDAC binding from opening areas of active euchromatin. On the other hand, short-term PEITC treatment had very minor impacts on the distribution of histone epigenetic regulators in SW620 cells, suggesting that long-term exposure is required to generate persistent and heritable alterations in tumor cell epigenetic profiles [244]. In human colon carcinoma cell line HT29, PEITC demonstrated a cytotoxic effect with an IC 50 of 11.88 µM [245]. Further in vivo investigation using PEITC indicated that a daily dose of 60 mg/kg p.o for 21 days could inhibit HDAC overexpression in rats [245]. Several HDACs, including HDAC1, HDAC3, SAP18 (sin3-associated protein 18), and SAP30 (sin3-associated protein 30), showed preferential attachment to heterochromatin after exposure to PEITC, indicating that PEITC may prevent HDAC binding from opening areas of active euchromatin. On the other hand, short-term PEITC treatment had very minor impacts on the distribution of histone epigenetic regulators in SW620 cells, suggesting that long-term exposure is required to generate persistent and heritable alterations in tumor cell epigenetic profiles [244]. In human colon carcinoma cell line HT29, PEITC demonstrated a cytotoxic effect with an IC50 of 11.88 μM [245]. Further in vivo investigation using PEITC indicated that a daily dose of 60 mg/kg p.o for 21 days could inhibit HDAC overexpression in rats [245].

Sulforaphane (SFN)
Sulforaphane (1-isothiocyanato-4-(methylsulfinyl)butane) ( Figure 5) is an isothiocyanate mainly distributed in diverse cruciferous vegetables, such as broccoli, cabbage, brussel sprouts, and radish [246]. Treatment with SFN (15 µM and 20 µM) for six days decreased the growth of MCF10A cells, suggesting that these concentrations may be hazardous to normal breast cells. Breast cancer cells are preferentially inhibited at doses of 10 µM or less. Sulforaphane therapy at a dose of 10 µM was shown to dramatically suppress HDAC activity in breast cancer cells [247]. Similar events were also noticed in A549 and H1299 lung cancer cells, where SFN significantly inhibited HDAC activity and upregulated the expression levels of acetylated histones H3 and H4 [248]. On the other hand, SFN increased the anti-oncogene proteins DUSP4 and CDK expression, which correlated with the downregulation of HDAC5 and HDAC11 genes in the hepatocarcinoma HepG2 cell line [249]. Listed in Table 5 are natural compounds that target HDAC in human cancers. Reduced CpG methylation in the miR-9-3 promoter region. Increased H3K4me1 enrichment at the miR-9-3 promoter. Induced the expression of miR-9-3. Reduced the epigenetic modifying enzymes' expression and activity.

Resveratrol
Resveratrol (3,5,4 -trihydroxystilbene) ( Figure 5) is a bioactive compound, first extracted from the roots of white hellebore (Veratrum grandiflorum Loes.) by Saiko et al. [276]. This bioactive compound is found in more than 50 different plant species, including grapes, apples, blueberries, plums, and peanut. It has been extensively explored for its health benefits against diverse pathologies, including cancer [277]. The cytotoxic evaluation of resveratrol against HeLa, SiHa, and Caski cells revealed that the IC 50 values were 17, 22, and 118 µM, respectively [259]. For Caski cells, treatment with resveratrol for four days increased p21 expression by 1.7-, 2.1-, and 28-fold, respectively, compared to control cells (HDAC inhibition is characterized by a rise in p21, which is controlled by UHRF1) [259].
The translocation of B16F10 tumor cells to the lungs was dramatically reduced after 20 days of intraperitoneal (50 mg/kg) resveratrol therapy in 7-week-old female mice. Resveratrol showed no cytotoxicity against B16F10 cells at a dose of 60 µM, and it was speculated that resveratrol is involved in its anti-migration function, since HDAC1 and ACAT1 are resveratrol targets [260]. Similarly, resveratrol treatments, even at low doses (10 µM), reduced the cell viability of MCF-7 and MDA-MB-231 cell lines in a dose-dependent manner. Using increasing amounts of resveratrol (0-100 µM), researchers found that it suppresses HDAC activity in both cell lines in a dose-dependent manner [278].

Curcumin
Curcumin ( Figure 5) has been reported to be a potent epigenetic regulator that has multiple effects on HDAC expression and activity. Curcumin downregulated the expression levels of HDAC1, HDAC3, and HDAC8 proteins and histone acetyltransferase p300, increasing the Ac-histone H4 protein expression in Raji cells. Curcumin was used at different concentrations (6.25, 12.5, 25, 50, and 100 µmol/L). Compared to curcumin-treated cells (25 µmol/L for 24 h), the expression of HDAC1, HDAC3, and HDAC8 was considerably higher in Raji cells. When cells were treated with 25 µmol/L curcumin for 24 h, more than two-thirds of them could not multiply. The IC 50 for 24 h was 25 µmol/L [263]. Curcumin was found to inhibit histone acetyltransferase (HAT) activity in LNCaP cells at a concentration of 5 µM, and it has also been suggested to be a possible DNMT and HDAC inhibitor [267].

Calebin-A
Calebin-A ( Figure 5) is a curcuminoid compound obtained from Curcuma longa roots. This molecule possesses various biological properties [279]. It inhibited the proliferation of the malignant peripheral nerve sheath tumor (MPNST (STS26T, ST8814, T265, and S462TY)). At concentrations of 12.5-25 µM, Calebin-A reduced the viability of MPNST cell lines. With a 25 µM dose of Calebin-A applied for 24 h; all MPNST cell lines had survival rates below 50%. On the other hand, HDAC activity in MPNST cells did not decrease significantly after treatment [268].

Pterostilbene
Pterostilbene (3,5-dimethoxy-4-hydroxystilbene) ( Figure 5) is a naturally occurring bioactive compound found in grapes and numerous berries, especially blueberries. Chemically, pterostilbene is a phytoalexin dimethyl ether compound derived from resveratrol [280]. Combinatorial treatment of pterostilbene with resveratrol (5 µM) modulated gene expression in HCC1806 and MDA-MB-157 breast cancer cells through epigenetic processes, including modification of HDACs [269]. It induced a downregulation of SIRT1, a type III HDAC, which is involved in modifying histones and some non-histone proteins via deacetylation, and subsequently modulates cell proliferation, apoptosis, stress response, metabolism, cellular senescence, and tumorigenesis [269,281]. concentrations, guggulsterone induced a remarkable effect on the modulation of key epigenetic regulators, such as HDACs responsible for the activation of tumor suppressor genes. Moreover, it has been reported that this bioactive compound can reverse the epigenetic modifications resulting from DNA hypermethylation, via suppressing HDAC1 expression in both human breast cancer MCF7 and MDA-MB-231 cell lines.

Withaferin A (WFA)
Withaferin A ( Figure 5) is a steroidal lactone extracted from the Withania somnifera, known for its antitumor properties, and targeting different hallmarks of cancer, including cell proliferation, migration, invasion, and angiogenesis, as well as the epigenetic process. WFA demonstrated chemopreventive effects against breast cancer, reversing the epigenetic changes via downregulation of HDAC1 protein levels in MCF7 and MDA-MB-231 cell lines. The IC 50 values for MCF 7 and MDA MB 231 cells were 8 and 10 µM, respectively. Downregulation was also observed in HDAC1 expression in MDA MB 231 cells. In another study, WFA alone or combined with SFN (at 1 and 5 µM, respectively) exhibited remarkable downregulation of HDAC1 expression at both mRNA and protein levels in the MCF7 and MDA-MB-231 cells, with a significant effect using combination therapy [250]. WFA with SFN also reduced HMT activities; however, they increased HAT activities, especially against MDA-MB-231 cells [250].
4.9. Phenolic Acids, Secoiridoids, Tannins, and Tanshinones 4.9.1. Phenolic Acids: Caffeic Acid Chlorogenic Acid Caffeic acid and its derivative chlorogenic acid ( Figure 5) are phenolic acids mainly found in numerous plants and have shown promising health benefits against several diseases. These two compounds are effective epigenetic agents, inhibiting HDAC activity. Using an in vitro HDAC assay, Bora-Tatar and co-workers [265] showed that chlorogenic acid exhibits potent inhibition of HDAC activity (IC 50 = 375 µM). In contrast, moderate inhibition was observed by caffeic acid (IC 50 = 2.54 mM).

Secoiridoids: Oleacein
Oleacein ( Figure 5) is the most abundant phenolic compound of Olea europaea L. (olive), belonging to the secoiridoid class. This compound has shown to be an efficient epigenetic modulator on multiple myeloma cell lines (NCI-H929, RPMI-8226, U266, MM1s, and JJN3). In fact, oleacein was shown to reduce cell viability in a dose-dependent manner, 48 h after treatment, with IC 50 s ranging from 5.0 to 20.0 µM. On the other hand, this molecule did not affect the vitality of PBMCs from healthy donors. Unlike the positive controls, TSA and SAHA, incubation with oleacein had no effect on the HDAC activity retrieved from nuclear extracts. These data imply that oleacein does not affect MM cell acetyloma through enzymatic HDAC suppression [270].

Secoiridoids: Ellagic Acid
Ellagic acid (EA) ( Figure 5) is a ubiquitous phenolic compound extracted from various fruits and vegetables, and which is well-known for its bioactivity against different cancer cells. This compound has demonstrated effective epigenetic HDAC modification. A significant increase in the expression of the HDACs gene was observed in human adipogenic stem cells treated with EA (twenty-fold higher than the control). Furthermore, 10 µM EA inhibited HDAC9 downregulation after four days of cell treatment. This phenolic compound also reduced adipocyte development and differentiation by inducing histone arginine methylation, and thereby increased acetylated histone through epigenetic changes, mediated by coactivator-associated arginine methyltransferase 1 (CARM1) inhibition. The findings indicated that CARM1 inhibition induces suppression of H3R17 methylation, resulting in decreased H3K9 acetylation and HDAC9 dissociation [271]. 4.9.4. Tanshinones: Tanshinone IIA Tanshinone IIA ( Figure 5) is a natural bioactive compound identified in the rhizome of Salvia miltiorrhiza Bunge. Wang et al. [272] investigated the role of tanshinone IIA in epigenetic pathways, illustrating its effect on HDAC modification. These researchers showed that this bioactive compound inhibited the enzymatic activity of HDACs by 50% in the tanshinone treatment concentration range of 5.0 to 10.0 µM. Tanshinone IIA also downregulated, in a concentration-dependent manner, HDAC1, HDAC3, and HDAC8 protein levels, by decreasing mRNA expression [272]. Arsenic trioxide (As 2 O 3 ), a naturally occurring inorganic compound and environmental pollutant, is considered a potent human carcinogen [273,282]. This compound has demonstrated an effective anticancer activity, acting on epigenetic regulators (Table 5); it increased HDAC4 expression in human HeLa and HEK293T cell lines [273]. Treatments conducted for 24-72 h in the concentration range of 0.2-0.8 µM showed that As 2 O 3 induced histone H4K16 acetylation in a dose-dependent manner. Arsenic trioxide downregulated the global histone H4 acetylation at lysine 16 (H4K16ac) via direct binding to histone acetyltransferase human male absent on first (hMOF) in HeLa and HEK293T cell lines. Additionally, HAT in vitro assay showed that arsenic trioxide directly inhibits hMOF activity [273].

Curcumol
Curcumol ( Figure 5) is a natural sesquiterpenoid isolated from numerous plants of the Zingiberaceae family and has demonstrated diverse biological properties, such as being anti-inflammatory, anti-proliferative, antioxidant, and antimicrobial [283]. Several research investigations have proven the potent anticancer activity of curcumol; targeting several checkpoints, including HDAC epigenetic mechanism. Curcumol has been shown to suppress choriocarcinoma cancer stem-like cell self-renewal (CSLCs) through HDAC activity (in vitro and in vivo). JEG cells treated with curcumol (75 µg/mL) for sphere formation showed 50% growth inhibition [274]. HDAC enzyme inhibition was assessed using a colorimetric kit assay after seven days of curcumol treatment.

Selenium
Research findings demonstrated that selenium is a potential chemopreventive agent against prostate cancer. In this line, Xiang et al. [275] studied the effect of a single dose of selenite (1.5 µM) for 7 days on the human prostate cancer cell line LNCaP. They reported that selenite could prevent prostate cancer through an epigenetic mechanism by maintaining the expression of GSTP1 promoter, inducing the inhibition of DNA and histone methylation, and enhancing histone acetylation. In addition, selenite treatment altered HDAC expression and significantly affected its activity. It decreased the expression levels of methylated histone H3 on lysine 9 (H3-K9) and increased the expression of acetylated H3-K9 [275].

Conclusions
In summary, this work has highlighted the close relationship between the enzymes that regulate chromatin architecture and tumor transformation. Indeed, HDACs are responsible, along with other epigenetic modifiers, for maintaining cellular memory during mitotic division. Disturbances of HDAC activities correlate with tumor transformation in several cancers. Through our bibliographic research, we have shown that natural bioactive substances can modify the enzymatic activity of HDAC, and, therefore, its regulation. These remarkable results demonstrate the possible pharmacological effects of these substances and their applications in cancer chemotherapy. However, further testing needs to be performed on these bioactive compounds, to first confirm their safety by investigating their toxicity, and with other in vivo tests, to assess their pharmacokinetics. The validation of these approaches could pave the way for the clinical use of some of these molecules against cancer.