Histone Deacetylase Inhibitors

International Journal of
Molecular Sciences
Review
Histone Deacetylase Inhibitors as Anticancer Drugs
Tomas Eckschlager 1, *, Johana Plch 1 , Marie Stiborova 2 and Jan Hrabeta 1
1 Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine,
Charles University and University Hospital Motol, V Uvalu 84/1, Prague 5 CZ-150 06, Czech Republic;
plchova.johana@gmail.com (J.P.); janhrabeta@gmail.com (J.H.)
2 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030/8,
Prague 2 CZ-128 43, Czech Republic; stiborov@natur.cuni.cz
* Correspondence: tomas.eckschlager@lfmtol.cuni.cz; Tel.: +42-060-636-4730
Received: 14 May 2017; Accepted: 27 June 2017; Published: 1 July 2017
Abstract: Carcinogenesis cannot be explained only by genetic alterations, but also involves epigenetic
processes. Modification of histones by acetylation plays a key role in epigenetic regulation of gene
expression and is controlled by the balance between histone deacetylases (HDAC) and histone
acetyltransferases (HAT). HDAC inhibitors induce cancer cell cycle arrest, differentiation and cell
death, reduce angiogenesis and modulate immune response. Mechanisms of anticancer effects
of HDAC inhibitors are not uniform; they may be different and depend on the cancer type,
HDAC inhibitors, doses, etc. HDAC inhibitors seem to be promising anti-cancer drugs particularly
in the combination with other anti-cancer drugs and/or radiotherapy. HDAC inhibitors vorinostat,
romidepsin and belinostat have been approved for some T-cell lymphoma and panobinostat for
multiple myeloma. Other HDAC inhibitors are in clinical trials for the treatment of hematological
and solid malignancies. The results of such studies are promising but further larger studies are
needed. Because of the reversibility of epigenetic changes during cancer development, the potency
of epigenetic therapies seems to be of great importance. Here, we summarize the data on different
classes of HDAC inhibitors, mechanisms of their actions and discuss novel results of preclinical and
clinical studies, including the combination with other therapeutic modalities.
Keywords: histone deacetylases; histone deacetylase inhibitors; cancer; apoptosis; autophagy;

cell cycle arrest; anti-angiogenic effect; drug combinations
1. Introduction
Cancer chemotherapy has been one of the major medical advances in the last few decades.
However, the drugs used for this therapy have a narrow therapeutic index, and the produced responses
are often only palliative as well as unpredictable. Such approaches, although directed toward certain
biomacromolecules, do not discriminate between rapidly dividing non-malignant and cancer cells.
In contrast, targeted therapy that has been introduced in recent years is directed against cancer-specific
targets and signaling pathways, and thus has more limited nonspecific mechanisms.
A crucial role for epigenetic mechanisms in cancer development is demonstrated by a number
of studies. Carcinogenesis cannot be explained only by genetic alterations, but also involve
epigenetic processes (DNA methylation, histone modifications and non-coding RNA deregulation).
Histone modifications include H3 and H4 histones lysine deacetylation that leads to chromatin
decondensation [1]. These alterations influence gene transcription including upregulation of several
anti-oncogenes and DNA repair genes [2]. Thus, the epigenetic processes have emerged as novel
therapeutic targets in numerous investigations.
The importance of histone deacetylase (HDAC) enzymes in organisms has been demonstrated
using the studies with mice knocked out on members of class I HDACs. HDAC1-null mice die
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Int. J. Mol. Sci. 2017, 18, 1414 2 of 25
prenatally with severe proliferation defects and general growth retardation; HDAC2-null mice
die the first day after birth for cardiac malformations; and HDAC3-null mice die prenatally for
defects in gastrulation [3,4]. HDACs seem to be important for gene expression [5]. It has been
described several times that their levels vary greatly in cancer cells and differ according to the
tumor type. HDAC1 is highly expressed in prostate, gastric, lung, esophageal, colon and breast
cancers [6–8]. High levels of HDAC2 were found in colorectal, cervical and gastric cancers [9,10].
In addition, HDAC3 is overexpressed in colon and breast tumors [11], whereas HDAC6 is highly
expressed in mammary tumors, HDAC8 is overexpressed in neuroblastoma cells and HDAC11
mainly in rhabdomyosarcoma [12–14]. Increased expression of different HDAC and/or histone
hyperacetylation in different cancers is caused by different mechanisms which may affect effects
of individual HDAC inhibitors.
The in vitro analysis of different HDACs isoforms using siRNA (small interfering RNA) against
HDAC1, HDAC2 and HDAC3 on several ovarian carcinoma cells (SKOV3, OVCAR3, IGROV-1, ES-2,
TOV112D, A2780 and A2780/CDDP) showed that knockdown of HDAC1 inhibits proliferation and
tumorigenicity, while knockdown of HDAC3 reduces cell migration with an increase in E-cadherin.
On the contrary, knockdown of HDAC2 has no effect on proliferation and tumorigenicity, or cell
migration [15].
In this review, we describe different classes of HDAC inhibitors and mechanisms of their actions,
and discuss novel data found in several preclinical experiments and clinical studies. Moreover, we discuss
their combination with other therapeutic modalities, particularly with DNA-damaging compounds.
2. Acetylation and Deacetylation

Modification of histones by acetylation plays a key role in epigenetic regulation of gene expression
by changing the structure of chromatin and by modulating the accessibility of transcription factors to
their target DNA sequences [16]. Histone acetylation is enriched in transcriptionally active regions of
the genome, especially at proximal promoters and enhancers, and facilitates the binding of transcription
factors. Acetylation loosens contact between core nucleosome proteins and DNA, thereby making
transcription factor binding sites more accessible, recruitment of bromodomain proteins which
recognize acetylated lysine residues on the N-terminal tails of histones and are actively recruited to
enhancer and promoter regions [17]. The acetylation state of histones and other proteins is maintained
by histone acetyltransferases (HAT) and HDAC. HATs catalyze the transfer of an acetyl group from
acetyl-CoA to the ε-NH2 group of lysine residues in proteins, while HDACs remove it [12]. Acetylation
status of non-histone proteins modifies many cellular functions e.g., mRNA splicing, transport and
integrity; translation; activity, localization, stability and protein interactions [18]. Of the proteins whose
functions are modulated by HDACs, the p53 (tumor protein 53), RUNX3 (Core binding factor α3
subunit), STAT3 (Signal transduction and activation of transcription 3), β-catenin, estrogen receptor,
Myc (Avian myelocytomatosis viral oncogene homolog), EKLF (Erythroid Kruppel-like factor), GATA
family (GATA-binding factors), HIF-1α (Hypoxia-inducible factor 1α), MyoD (Myogenic regulatory
factor), NF-κB (Nuclear factor κB) or Foxp3 (Forkhead box P3 protein), are the most important in
cancer (for review, see [19]).
Based on the homology to their yeast analogs, HDACs are divided into four classes. Class I,
located in the nucleus, includes HDACs 1, 2, 3 and 8. HDACs 4, 5, 7 and 9 are members of class IIa,
and class IIb (isoforms 6 and 10) are located in both cytoplasm and nucleus. Class IV (only the isoform
HDAC11) exhibits some features of classes I and II. NAD+ -dependent homologs 1–7 of the yeast Sir2
proteins (sirtuins) are designed as the class III of HDACs [20]. To remove the acetyl groups of proteins,
the HDACs utilize two different mechanisms. These mechanisms act as the base for their classification
into two distinct families. The “classical family” comprises of Zn2+ -dependent HDACs (classes I, II and
IV). The Zn2+ ion stabilizes the acetylated substrate in the catalytic center of the enzyme and polarizes
the carbonyl group making the carbon to be a better target for nucleophilic water molecules [21].
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The further HDAC family is NAD+ -dependent, being capable of forming O-acetyl ADP ribose and
nicotinamide as a result of the acetyl transfer [22].
Most of the classic HDACs form large complexes with multiple transcriptional co-repressors
(such as Sin3 (Transcriptional regulatory protein SIN3), NuRD (Nucleosome remodeling and
deacetylase complex), NcoR (Nuclear receptor co-repressor) and SMRT (Silencing mediator of retinoic
acid and thyroid hormone receptors) with chromatin remodeling activity [23]. HDAC complexes
can contribute to gene repression through two mechanisms—specific targeting by repressors and the
constitutive association with chromatin [24,25].
HATs, which have the counterbalancing effect of HDAC, are a diverse set of multi-subunit
complexes. They are divided into Gcn5 N-acetyltransferases (GNATs) and MYST HATs. GNATs include
Gcn5 (General control non-repressed protein 5), PCAF (p300-CREB-binding protein-associated factor),
Elp3 (Elongator protein 3), Hat1 (Histone acetyltransferase 1), Hpa2 (Hrp-associated 2 protein) and
Nut1 (Component of the RNA polymerase II mediator complex). MYST is an acronym for the members
of a protein family: Morf, Ybf2 (Sas3), Sas2 and Tip60. Although these two groups of HATs are the
main ones, other proteins such as p300/CBP (CREB-binding protein), Taf1 (TATA box-binding protein
associated factor 1) and a number of nuclear receptor coactivators show intrinsic acetylase activity.
However, they do not contain true consensus HAT domains and therefore represent an “orphan class”
of HAT enzymes [26].
3. HDAC Inhibition and Its Effects

Histone acetylation has been shown to be an important regulatory mechanism that controls
transcription of approximately 2–10% of genes [27]. Deacetylation of histones causes chromatin
condensation, while decondensation is caused by increased acetylation [1]. Such changes might
result in decreased or increased gene transcription. However, there are other proteins than histones
(e.g., chromatin remodeling proteins, DNA-binding nuclear receptors, DNA repair enzymes, signaling
mediators, structural proteins, transcription coregulators, DNA-binding transcription factors) whose
activity is affected by acetylation. In particular, the function of transcription factors can be positively or
negatively affected. It provides an explanation why gene expression determined by HDAC inhibition,
is not always increased even if the chromatin structure is loosened. The changes in HAT/HDAC activity
balance can: (I) lead to an altered gene expression profile as well as to the change of some signaling
pathways e.g., ERK (Extracellular signal-regulated kinase) and Wnt (Wingless/Int-1) pathways;
(II) affect proteasomal degradation; (III) influence protein kinase C activity and (IV) change DNA
methylation status. In various cancer cells, the shift to an increased acetylation/deacetylation ratio by
HDAC inhibition was found to have a substantial effect on their fate (for overview see [19]).
HDAC inhibitors may act specifically against the only several types of HDACs
(HDAC isoform-selective inhibitors), but also against all types of HDACs (pan-inhibitors).
HDAC inhibitors can be classified as members of five classes of compounds: (I) hydroxamic acids
(hydroxamates); (II) short chain fatty (aliphatic) acids; (III) benzamides; (IV) cyclic tetrapeptides;
and (V) sirtuin inhibitors including the pan-inhibitor nicotinamide and the specific SIRT1 and SIRT2
inhibitors sirtinol and cambinol, respectively (for overview, see [28] and Table 1).
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Table 1. Overview of selected histone deacetylases (HDAC) inhibitors.
Class HDAC Inhibitor Target HDAC Class Clinical Status

Trichostatin A pan preclinical
SAHA pan approved for cutaneous T-cell lymphoma
Belinostat pan approved for peripheral T-cell lymphoma
Panabiostat pan approved for multiple myeloma
Givinostat pan phase II clinical trials—relapsed leukemia and multiple myeloma
hydroxamic acids Resminostat pan phase I and II clinical trials—hepatocellular carcinoma
Abexinostat pan phase II clinical trial—B-cell lymphoma
Quisinostat pan phase I clinical trial—multiple myeloma
Rocilinostat II phase I clinical trial—multiple myeloma
Practinostat I, II and IV phase II clinical trial—prostate cancer
phase I clinical trial—advanced/metastatic solid tumors refractory to
CHR-3996 I
standard therapy
approved for epilepsia, bipolar disorders and migraine, phase II clinical
Valproic acid I, IIa
short chain fatty trials—several studies
acids Butyric acid I, II phase II clinical trials—several studies
Phenylbutyric acid I, II phase I clinical trials—several studies
phase II clinical trials—breast cancer, Hodgkin´s lymphoma, non-small cell
Entinostat I lung cancer, phase III clinical trial—hormone receptor positive
breast cancer
benzamides
Tacedinaline I phase III clinical trial—non-small cell lung cancer and pancreatic cancer
4SC202 I phase I clinical trial—advanced hematological malignancies
Mocetinostat I, IV phase II clinical trials—Hodgkin´s lymphoma
cyclic
Romidepsin I approved for cutaneous T-cell lymphoma
tetrapeptides
Nicotinamide all class III phase III clinical trial—laryngeal cancer
Sirtinol SIRT 1 and 2 Preclinical
sirtuins inhibitors
Cambinol SIRT 1 and 2 Preclinical
cancer preclinical, phase I and II clinical trials—Huntington
EX-527 SIRT 1 and 2
disease, glaucoma
(I) Whereas trichostatin A (TSA) is an HDAC inhibitor used only in laboratory experiments
because of its toxicity, vorinostat (suberoylanilide hydroxamic acid, SAHA) which has been already
approved by United States Food and Drug Administration (FDA) as the first HDAC inhibitor, is utilized
for the treatment of relapsed and refractory cutaneous T-cell lymphoma (CTCL). Belinostat (PXD-101)
(approved for therapy of peripheral T cell lymphoma (PTCL)), panobinostat (LBH589) (approved for
therapy of multiple myeloma) and the compounds recently tested in clinical studies, i.e., givinostat
(ITF2357), resminostat (4SC201), abexinostat (PCI24781) and quisinostat (JNJ-26481585), are the
pan-HDAC inhibitors. Selective HDAC inhibitors in the group of hydroxamic acids, including
rocilinostat (ACY1215), which is selective inhibitor of HDAC class II, as well as practinostat (SB939)
that inhibits all classical classes of HDACs (I, II and IV) and CHR-3996, the selective inhibitor of class I,
are under clinical studies.
(II) The short chain fatty acids, valproic acid (VPA), butyric acid and phenylbutyric acid, are known
to be weak inhibitors of HDAC class I and IIa and I and II, respectively. VPA is registered for the
therapy of epilepsy, bipolar disorders and migraines, and is now together with other short chain fatty
acids HDAC inhibitors tested in clinical studies as anticancer drugs.
(III) Among the benzamides that are now tested in clinical studies, entinostat (MS-275-SNDX-275),
tacedinaline (CI994) and 4SC202 inhibit the class I HDACs. Mocetinostat (MGCD0103) is a selective
inhibitor of classes I and IV HDACs.
(IV) The cyclic tetrapeptides include the bicyclic depsipeptide romidepsin (FK228, FR901228),
a compound that has been approved by FDA and EMA (European Medicines Agency) to treat CTCL.
It is a prodrug, which is reductively activated to a metabolite containing a thiol group that chelates the
zinc ions in the active center of the HDAC of class I [29].
(V) Sirtuin inhibitors include the pan-inhibitor nicotinamide and the specific SIRT1 and SIRT2
inhibitors sirtinol, cambinol and EX-527. They might act against different types of neurodegenerations
and cancers [30].
The other different HDAC inhibitors and the mechanisms of their actions are reviewed
elsewhere (see [31–37]). Many various compounds with HDAC inhibiting activity are now being
preclinically investigated.
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4. Mechanisms of HDAC Inhibitors Action

HDAC inhibitors induce cancer cell cycle arrest, differentiation and cell death. Moreover, they reduce
angiogenesis and modulate immune response. Hypothesis of “epigenetic vulnerability of cancer cells”,
which has been proposed by Dawson and Kouzarides [38], is a cause of relative specificity of HDAC
inhibitors. This hypothesis supposed that normal cells have in contrast to some cancer cells multiplied
epigenetic regulatory mechanisms. Therefore, HDACs may be essential for the maintenance of a set of
key genes required for survival and growth of cancer cells but not of normal ones [38].
Mechanisms of anticancer effects of HDAC inhibitors are not uniform; they may be different
and depend on a type of cancer, on the individual HDAC inhibitor and its dose as well as on some
other factors [39]. For example, VPA has shown to inhibit the invasiveness in bladder cancer but not in
prostate cancer cells [40], while it has not induced cell cycle inhibition in some neuroblastoma cell lines
such as SH-SY5Y and SK-N-BE [41].
4.1. Cell Cycle Arrest

Cell cycle arrest induced by HDAC inhibitors is caused by several mechanisms; the most important
seems to be the increased expression of cell cycle genes such as CDKN1A (Cyclin dependent kinase
inhibitor p21) as described in a variety of cancer cells [42–44]. Its product blocks the formation
of dimers from cyclins and cyclin dependent kinases (CDKs) that induce cell cycle arrest and
inhibition of cell differentiation [43,44]. The expression of p21 is modulated by protein p53 that
interacts with p21 promoter, competing with HDAC1, which decreases transcription of p21 [45].
After treatment with HDAC inhibitors, the HDAC1 protein is released from the Sp1 (Promoter-specific
RNA polymerase II transcription factor), which increase p21 expression. Furthermore, HDAC
inhibition increases acetylation of the p53 protein which results in an increase in its half-life [46],
thereby improving the interaction with the p21 promoter [47]. Moreover, the p53 protein interactions
with its activators ASPPs (Ankyrin-repeat-, SH3-domain- and proline-rich region containing proteins),
53BP1 (p53-binding protein), TiP60/hMOF (Human males absent on the first), hCAS/CSE1L (Cellular
apoptosis susceptibility protein), and HZF (Hematopoietic zinc finger) are regulated by its acetylation
status which is influenced by HDAC inhibitors [48]. Finally, the p21 levels are increased, thereby
mediating cell cycle arrest and apoptosis [43,49,50]. HDAC inhibitors can also inhibit expression
of genes coding cyclin D and cyclin A resulting in the absence of activities of the corresponding
kinases, CDK2 and CDK4 [44,51]. In addition, the HDAC inhibitors may increase the stability and
transcriptional activities of RUNX3, which mediates induction of p21 and product of anti-apoptotic
gene Bim (Bcl-2-interacting mediator of cell death) [52–55].
4.2. Apoptosis Induction

HDAC inhibitors induce apoptosis in tumor cells by regulation of pro-apoptotic and anti-apoptotic
genes (for a review see [56–58]). The mechanisms by which different HDAC inhibitors induce apoptosis
include activation of both extrinsic and intrinsic apoptotic pathways.
Initiation of the extrinsic apoptotic pathway by HDAC inhibitors was proven in many
in vitro experiments. HDAC inhibitors have been demonstrated to influence death receptors TRAIL
(TNF related apoptosis inducing ligand), DR5 (Death receptor 5), Fas (TNF superfamily 6), TNF
(Tumor necrosis factor) and TNF-related ligands Fas-L, LIGHT (TNF superfamily member 14) and TLA1
(Transparent leaf area peptide). Inhibition of those death receptors and their ligands inhibits apoptosis
induced by HDAC inhibitors [57,59–61]. In vivo experiments with xenograft using tumor cells with
TRAIL and Fas suppressed by siRNA showed a significant decrease in apoptosis after treatment with
VPA [62]. HDAC inhibitors also activate intrinsic apoptotic pathway. They regulate transcription of
pro-apoptotic genes such as Bid (BH3 interacting domain death agonist protein), Bad (Bcl-2 associated
agonist of cell death protein) and Bim that activate the intrinsic apoptotic pathway [42,58,63,64].
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It can be concluded that in tumor cells exposed to HDACs inhibitors pro-apoptotic genes involved
in the extrinsic (TRAIL, DR5, FAS, FAS-L, and TNF-α) and/or intrinsic apoptotic pathways (BAX, BAK
and APAF1) are up-regulated, while anti-apoptotic genes (Bcl-2 and XIAP (X-linked inhibitor of
apoptosis protein)) are downregulated [10]. HDAC inhibitors can, however, enhance the levels of
anti-apoptotic protein Bcl-2 via activation of ERK [65]. Besides, these effects on gene expression,
the HDAC inhibitors increase amounts of reactive oxygen species (ROS) that can induce apoptosis
in leukemic cells (Jurkat, ML-1, U937, HL-60, K-562, CEM-CCRF and its doxorubicin selected
P-glycoprotein overexpressing subline and FDC-P1 and sublines overexpressing Bid, Bcl-2 and Bid
plus Bcl-2) [63,66,67]. SAHA and entinostat increase expression of binding protein-2 (TBP-2) that
inhibits thioredoxin in LNCaP prostate cancer, T24 bladder cancer and MCF7 breast cancer cells [68].
Thioredoxin is an intracellular antioxidant, therefore treatment of tumor cells by these HDAC inhibitors
induce ROS-dependent apoptosis [69,70]. We found that VPA induces apoptosis more effectively
under hypoxic conditions and overcomes hypoxia-induced resistance to cisplatin (CDDP) in high risk
neuroblastoma derived cells UKF-NB-3 and CDDP resistant subline [71] probably by induction of
HIF-1α degradation [72].
4.3. The Effects on Induction of Autophagy

Acetylation of many autophagy-related proteins, such as the product of the autophagy-related
genes (ATGs), is regulated by the balance between HATs and HDACs [73]. Autophagy can also
be regulated through the acetylation of transcription factors such as FOXO [74]. Different HDACs
influence autophagic activity by different mechanisms as showed the results of several experiments
described below. HDAC6 induces autophagy when the ubiquitin-proteasome system (UPS) is impaired.
Knockdown of HDAC2 inhibits autophagy in cardiomyocytes [75]. On the contrary, inhibition or
knockdown of HDAC1 in HeLa cells promote formation of autophagic vacuoles [76]. HDAC10 induces
autophagy-mediated cell survival in neuroblastoma (E(2)-C, Kelly, and IMR32 cells) and its inhibition
sensitizes cells to cytostatics [77]. SIRT1 forms a complex with components of the autophagy machinery
(Atg5, -7, and -8) and stimulates autophagy [78].
The role of HDAC inhibitors induced autophagy in the death of cancer cells is still controversial.
Some studies have reported that autophagy serves as a cell death mechanism as either autophagy
inhibitors or knockdown of ATGs reduces the anticancer effect of HDAC inhibitors. Moreover, using
different in vivo models, a combination of HDAC inhibitors and autophagy inhibition reduces colon
cancer cells HCT116 growth [79]. In contrast, autophagic degradation of intracellular components is
a death signal and leads to the cytotoxic effect of autophagy. For example, in panel of hepatocellular
carcinoma cells SAHA-induced cytotoxicity is inhibited by 3-methyladenine, which inhibits autophagy
by blocking the autophagosome formation via the inhibition of class III PI3K, or by Atg5 knockout [80].
Cell death in endometrial stromal sarcoma cells induced by SAHA is caused by autophagy [81].
SAHA induces apoptosis in TP53 wild type cancer cells, while the absence or degradation of
cytoplasmatic p53 leads to activation of the autophagic pathway which consequently induces cell
death [82]. The above-mentioned discrepancies might be due to differences in the used models, cancer
cells, HDAC inhibitors and their doses.
Several signaling pathways play a role in the induction of autophagy by HDAC inhibitors.
mTOR (Mechanistic target of rapamycin) is one of the most important suppressors of autophagy via
phosphorylation and inactivation of the ULK1 (Unc 51 like autophagy activating kinase 1) complex
which is an upstream component of the autophagy pathway. mTOR inactivation by SAHA restores
functions of ULK1 [80,83,84]. Overexpression of ATG genes induced by SAHA is caused by the
stimulation of NF-κB activity via modulation of RelA/p65 (NF-κB p 65 subunit) signaling [85].
Some studies also show that SAHA cause autophagy by ROS production in leukemic and hepatocellular
carcinoma derived cells [84,86]. The fact that some HDAC inhibitors can induce cell death in the
apoptosis-resistant cells by induction of autophagy seems to be the important feature for clinical
practice. Romidepsin and HDAC1 siRNA induce autophagy in HeLa cells [76]. SAHA inhibits
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growth of short term culture glioblastoma cells xenografts in nude mice by autophagy induction via
downregulation of AKT-mTOR signaling [87]. Based on the above-mentioned facts, we can suppose
that induction of autophagy by HDAC inhibitors may be a promising therapeutic anticancer strategy.
4.4. The Effects on Non-Coding RNA

HDAC inhibitors have been described to alter non-coding RNA expression. TSA and SAHA induce
miR-129-5p overexpression and apoptosis in thyroid cancer cells. miR-129-5p alone induces cell death
and knockdown experiments showed that its expression is necessary for HDAC inhibitor-induced
cell death [88]. Treatment of breast cancer cells and normal human fibroblasts with a combination
of sodium butyrate and panobinostat increased the expression of miR-31 which induces cellular
senescence via inhibition of BIM1 (Bisindolylmaleimide-based protein kinase C inhibitor 1) [89].
This was demonstrated by transfection of cells by vector overexpressing BMI1 and on the other hand
by its knock down by BMI1 shRNA. HDAC inhibition in B-cell lymphomas cells (Daudi, Ramos, Raji,
Su-DHL-6, OCI-Ly-19 and OCI-Ly-3) and non-malignant B cells silences Myc-mediated transcriptional
repression of the miR-15 and let-7 miRNA families, which induces apoptosis by downregulation of the
anti-apoptotic genes Bcl-2 and Bcl-xL and, thus, activates apoptotic pathways [90]. HDAC1 enhances
miRNA processing by acetylating protein DGCR8 (DiGeorge critical region 8 protein) which processes
miRNA and this acetylation increases the production of mature miRNAs in embryonic kidney
cells and acute myeloid leukemia cells (AML) [91]. Moreover, several HDACs are also targeted
by miRNA, e.g., miR-449a regulates prostate cancer both androgen dependent and independent cells
(PC-3, DU-145, BHP-1 and LNCaP) growth and viability by targeting and repressing HDAC1 [92].
Long non-coding RNAs (lncRNAs) are important cellular regulator molecules at the
transcriptional and post-transcriptional level. They exhibit a wide spectrum of functions such as
regulation of alternative splicing, transcriptional patterns, and protein activity. They also have
epigenetic effects and are precursors of miRNAs [93]. One of the mechanisms of transcription regulation
by lncRNAs is recruiting histone modifying complexes (e.g., PRC (Polycomb repressive complex)) to
target loci which are either activated or silenced, depending on the histone marks [94].
The data of Yang et al. showed that approximately 5% of intergenic lncRNA and about 6% of
protein coding genes were significantly differentially expressed in hepatocellular cancer cell lines
(Huh7, Bel7402, Bel7721, and HepG2) after treatment with TSA [95]. Intergenic lncRNAs regulate gene
expression through modifying the chromatin complexes or RNA binding proteins and their aberrant
expression is associated with cancer [96].
Low expression of lncRNA Xist (X inactive specific transcript which plays a major role in the
X inactivation process) seems to be a predictor of efficacy of abexinostat in breast cancer cells [97].
One may speculate from the above-mentioned facts that changes of lncRNAs expression may be one of
the mechanisms of the anticancer effect of HDAC inhibitors. However, further studies with non-coding
RNAs are necessary.
4.5. The Effects on Cellular Signaling Pathways

Another mechanism of anticancer effects of HDAC inhibitors is regulation of cell differentiation
by activation of some of protein kinases (i.e., ERK). Protein kinases modulate biological processes like
cell growth, differentiation and apoptosis. HDAC inhibitors were found to increase DNA binding and
transactivation activity of AP-1 transcription factor via ERK activation, increase expression of c-Jun
(Jun activation domain binding protein) and induce its phosphorylation in SH-SY5Y neuroblastoma
cells [65,98]. Although it is not yet clear how HDAC inhibitors affect ERK, it is supposed that
they can induce synthesis of a still unknown factor that activate the ERK signaling pathway [65]
or is incorporated into phospholipid molecules that activates ERK via the phosphatidylinositol
3-kinase (PI-3K)/Janus kinase 2 (JAK 2)/MEK-1-dependent and the tyrosine kinase-Ras-dependent
pathways [99,100]. HDAC inhibitors also increase the expression of genes that are involved in
regulation of ERK/AP-1 signaling, for example the growth associated protein-43 (GAP-43) and Bcl-2,
Int. J. Mol. Sci. 2017, 18, 1414 8 of 25
and hence they might increase growth of some cancer cells, e.g., in SH-SY5Y neuroblastoma cells or
immature cortical neuroblasts in GAP-43−/− mice [65,101].
VPA also affects Wnt signaling that is due to phosphorylation of serine 9 in the glycogen synthase
kinase-3β (GSK-3β) [102]. The Wnt signaling pathway plays an important role in various cancers
such as colon, breast, ovarian, prostate and endometrial cancers as well as medulloblastoma and
melanoma [103]. Inactivation of APC (Adenomatous polyposis coli) functions and of β-catenin
induces overexpression of the HDAC2 which protects colorectal cancer cells HT-29 from death [10].
Therefore, we may speculate that HDAC2 is a target of APC/β-catenin. Moreover, HDAC inhibitors
decrease polyp generation in a colon carcinoma model of APC deficient mice, probably via damage
of the HDAC2 by degradation in proteasomes [10]. Furthermore, VPA increases proliferation and
self-renewal of normal hematopoietic stem cells by inhibition of GSK-3β that activates the Wnt
pathway [104].
Acetylation of p53, which may be caused by HDAC inhibitors, decreases generation of a complex
p53/Mdm2 E3 ligase, whereas hypoacetylation increases its degradation by the proteasome and cancels
p53-mediated growth arrest and apoptosis as demonstrated by experiments with human non-small
cell carcinoma cells H1299 transfected by different Tp53 mutants [48]. In addition, acetylation induced
by HDAC inhibitors influences the expression of several proteasomal enzymes (Ubc8 E2 ubiquitin
conjugase, RLIM-subunit of the SCF E3 family of the ubiquitin ligase) in human embryonal kidney
cells [105].
4.6. The Antiangiogenic Effect

HDAC inhibitors can affect cancer angiogenesis and inhibit cellular stress response pathways,
thereby interfering with the metastatic process. The anti-angiogenic effects are associated with
down-regulation of pro-angiogenic genes such as the genes of the vascular endothelial growth factor
(VEGF) and/or endothelial nitric oxide synthase (eNOS) [106–108]. Expression and enzymatic activity
of eNOS are influenced by its phosphorylation which is catalyzed by Akt and numerous hemodynamic
and hormonal stimuli [109–111]. HDAC inhibitors decrease the stability of eNOS mRNA by binding
to the 50 -untranslated region of eNOS mRNA [112]. In addition, HDAC inhibitors decrease the levels
of the VEGF receptors in neuroblastoma cells [113].
Moreover, HDAC inhibitors induce hyperacetylation of HIF-1α, a pro-angiogenic transcription
factor, which induce its degradation [72]. VPA decreases angiogenesis by enhancing production of the
anti-angiogenic proteins thrombospondin-1 and activin A via downregulation of pro-angiogenic factors
such as the basic fibroblast growth factor (bFGF) [114]. Expression array showed that in prostate cancer
cells VPA induces the up-regulation of TSP1 (Thrombospondin-1), multiple TIMP (Tissue inhibitor of
metalloproteinase) isoforms and TGFβ (Transforming growth factor β), while expression of IGF1 (Insulin like
growth factor 1) and VEGF is decreased [115]. VPA and TSA decrease the formation of capillary tubes
of HUVEC (human vascular endothelial cells), but they do not induce their apoptosis [116].
On the contrary, Lin et al. found that HDAC inhibition resulted in the development of metastatic
phenotype including increasing of matrix metalloproteinases (MMPs) expression in some cancer
cell lines (liver (SNU-398, Huh6, HepG2, Hep3B, Huh7, PLC5, HCC36, TONG, HA59T, Sk-hep-1,
HA22T, and Malhavu), lung (H358, H1437, H661, H226Br, H1299, CL1-3, CL1-0, H23, H928,
and A549), gastric (NUGC, SC-M1, AZ521, AGS, and HR), and breast (MDA-231, Hs578T, and MCF-7)
cancer) [117]. Degradation of extracellular matrix by MMPs is important for angiogenesis, tissue
invasion and metastasis.
4.7. HDAC Inhibitor-Induced Modulation of Immune Response

A decrease in HDAC activity alters expression of MHC (Major histocompatibility complex) and
costimulatory molecules [118,119]. The subsequent increase in immunogenicity intensifies activation
of T cells and the prolonged survival of experimental animals [120,121]. Moreover, inhibition of
HDAC6 activates naïve T cells [119,120]. In addition, HDAC inhibitors influence different lymphocyte
Int. J. Mol. Sci. 2017, 18, 1414 9 of 25
populations. Inhibition of Class II HDACs enhances number and function of Treg and Class I HDAC
inhibitors enhances the functions of NK cells and CD8 T cells [122].
After exposure to SAHA or entinostat breast (MDA-MB-231), prostate (LNCaP) and pancreas
(AsPC-1) carcinoma cells are more sensitive to T-cell-mediated lysis in vitro [123]. This increased
immune reaction is directed against the HLA (Human leukocyte antigens) class I/epitope complexes
and the increased sensitivity to antigen-specific cytotoxic T-lymphocyte lysis indicate that HDAC
inhibition induces immunogenic modulation by promoting a signature of immune recognition.
Several tumor associated antigens have been shown to be epigenetically silenced in malignancies
impede immune recognition by cytotoxic T cells and contributing to worse prognosis [124,125].
The exposure of human carcinoma cells to SAHA has previously been shown to result in upregulation
of HLA related genes [126].
4.8. The Effects on Stem Cells

Epigenetic changes are important for reprogramming of somatic cells into pluripotent stem
cells. Therefore, several inhibitors of epigenetic-modifying enzymes including HDAC are able
to reprogram somatic cells into the pluripotent stem cells by modifying a chromatin structure
and making it more permissive to transcription factors [127]. There was described amplification
and maintenance of normal human hematopoietic stem cells induced by HDAC inhibitors [128],
enhancement of the epithelial–mesenchymal transition of colorectal and breast cancer cells [129,130]
and induction of CD133 (a marker of cancer stem cells in some cancers including the brain tumors)
expression in human glioma [131]. The experimental in vitro study showed that HDAC3 promoted
self-renewal of glioma stem cells and those experimental results were proved also by the studies
utilizing the tumor samples [132]. Our recent study shows that HDAC inhibitors (VPA, SAHA,
and MS-275) increase CD133 significantly in neuroblastoma cell lines that do not show methylated
its CpG promoters and VPA treatment may increase CD133+ cells that show higher resistance
to cytostatics than CD133− cells. This increase, found in the CD133+ cells was not caused by
elimination of CD133− cells. Moreover, VPA treatment enhanced clonogenicity and the ability to
generate neurospheres, increased Akt phosphorylation and induced expression of the pluripotency
transcription factors (Oct-4 (octamer-binding transcription factor 4), Nanog (Homeobox Transcription
Factor Nanog), Sox2 (sex determining region Y)) [133]. Similar increase of CD 133+ cells was found
also in glioblastoma cells treated by VPA [134]. Amplification of cancer “stem like cells” might be
unwanted action of HDAC inhibitors.
4.9. Other Effects of HDAC Inhibitors

Several HDAC inhibitors are known to be metabolized by cytochrome P450 (CYP) and they also
influence expression of the CYP enzyme proteins and their enzyme activities. Since CYP enzymes are
involved in biosynthesis and metabolism of many endogenous physiologically active substances and
drugs, this mechanism may be involved in potentiating of the effects of some anticancer drugs by HDAC
inhibitors. We found that VPA and TSA change CYP enzyme expression in neuroblastoma cells [135].
Several proteins important for DNA repair e.g., Ku70, flap structure-specific endonuclease 1
(FEN1), Werner syndrome (WRN), ataxia teleangiectasia mutated protein (ATM), mediator of DNA
damage checkpoint 1 (MDC1) and DNA-dependent protein kinases (DNA PK) are regulated by
acetylation which may be increased by HDAC inhibitors [136]. HDAC1 and 2 inhibition decreases
the DNA repair processes mediated by BAL-associated protein (BBAP) which protects cells against
DNA-damaging agents [137]. HDAC6 and SIRT2 are able to deacetylate α-tubulin and so stabilize
microtubules [138].
HDAC inhibitors have also anti-parasitic activity, e.g., against Plasmodium and Trypanosoma [139].
Above mentioned mechanisms of HDAC inhibitors (see Figure 1 and Table 2) are involved in
different HDAC inhibitors and in different cancers to varying degrees.
Int. J. Mol. Sci. 2017, 18, 1414 10 of 25
HDAC inhibitors have also anti-parasitic activity, e.g., against Plasmodium and Trypanosoma
[139].
Above
Int. J. Mol. Sci. 2017,mentioned
18, 1414 mechanisms of HDAC inhibitors (see Figure 1 and Table 2) are involved in
10 of 25
different HDAC inhibitors and in different cancers to varying degrees.
Figure 1. Mechanism of anticancer effects of HDAC inhibitors. ROS: reactive oxygen species.
Figure 1. Mechanism of anticancer effects of HDAC inhibitors. ROS: reactive oxygen species.
Table 2. Main
Table mechanism
2. Main ofofanticancer
mechanism anticancereffects of HDAC
effects of HDACinhibitors—preclinical
inhibitors—preclinical studies.
studies.
HDAC
Mechanism
Mechanism Target
Target Model
Model HDAC InhibitorRef. Ref.
Inhibitor
CDKN1A/p21
CDKN1A/p21 in
in vitro
vitro leukemic
leukemic cells cellsU937
U937 SAHASAHA [42] [42]
CDKN1A/p21
CDKN1A/p21 in vitro
vitro bladder
bladder cancer cancercells
cellsT24
T24 SAHASAHA [43] [43]
in vitro
in vitro normal
normal breast breastMCF-10A,
MCF-10A,prostate
prostatecancer
cancerPC-3,
PC-3,
CDKN1A/p21
CDKN1A/p21 DU145, colon
DU145, colon cancer
cancer SW620,
SW620,ovarian
ovariancancer
cancerIGROV,
IGROV,breast FR901228
FR901228 [44] [44]
cancer MCF-7,MCF-7,
breast cancer lung cancer
lung A549
cancer A549
in vitro
in vitro colon
colon cancercancer HCT116
HCT116++ knockknockoutoutDNMT1
DNMT1−−/− ,,
/−
p53p53 −−/−/,−DNMT1−/−− −
TSA TSA [46] [46]
DNMT3B
DNMT3B , DNMT1 and
/ and DNMT3B /− HCT116
DNMT3B −
−/− HCT116
p53 in vitro lung cancer A549 depsipeptide [47]

Cell cycle p53 in vitro lung cancer A549 depsipeptide [47]
Cell cycle arrest in vitro lung cancer H1299, osteosarcoma U2OS, TSA,
arrest p53 in vitro lung cancer H1299, osteosarcoma U2OS, human [48]
p53 human embryonal kidney HEK293 nicotinamide
TSA, nicotinamide [48]
embryonal kidney HEK293
p21 in vitro colon cancer HCT116 + knock out p53−/−, p21−/− sodium butyrate [49]
p21 in
in vitro
vitro colon
gastriccancer cancerHCT116
TMK-1,+MKN-1,
knock out p53−/− , p21−/−
MKN-7, sodium butyrate [49]
p21 MKN-28, MKN-74, MKN-45, KATO III,
in vitro gastric cancer TMK-1, MKN-1, MKN-7, MKN-28, HSC-39, TSA [50]
p21 MKN-74, MKN-45,
oral squamous KATO III,
carcinoma HSC-39,
HSC-4, oral squamous
Ho-1-N-1, Ho-1-U-1 TSA [50]
carcinoma HSC-4, Ho-1-N-1,
in vitro pancreatic endocrineHo-1-U-1
tumors- insulinoma CM,
RUNX3 TSA [52]
carcinoid
in BON, somatostatinoma
vitro pancreatic endocrine tumors- QCP-1
insulinoma CM,
RUNX3 TSA [52]
RUNX3 carcinoid BON, somatostatinoma
in vitro prostate cancer DU-145, LNCaP,QCP-1 PC-3 TSA [55]
death
RUNX3 receptor in vitro
in vitro prostate
leukemiccancer cells HL60
DU-145, LNCaP, PC-3 apicidin
TSA [60] [55]
in vitro AML cells from patients, normal CD34+
death receptor
death receptor in vitro leukemic cells HL60 apicidin
MS275 [61] [60]
progenitors
death receptor in vitro AML cells from patients, normal CD34+ progenitors MS275 [61]
intrinsic pathway in vitro leukemic cells U937 SAHA [42]
intrinsic pathway in vitro
vitro leukemic
T cell leukemia cells U937
CEM-CCRF and doxorubicin SAHA [42]
Apoptosis intrinsic pathway SAHA [63]
derived
in vitro TP-gp+(CEM-P-gp)
cell leukemia CEM-CCRF and doxorubicin
intrinsic pathway SAHA [63]
in vitro fetal
derived lung fibroblasts IMR90, osteosarcoma
P-gp+(CEM-P-gp)
intrinsic pathway SAHA, TSA [64]
U2OS
in vitroand
fetalSaos-2, colon cancer
lung fibroblasts HCT116
IMR90, p53−/−
osteosarcoma U2OS
intrinsic pathway SAHA, TSA [64]
Apoptosis intrinsic pathway in vitro
and leukemic
Saos-2, cells Jurkat,
colon cancer HCT116 p53−/−
ML-1 MS275 [66]
intrinsic pathway
intrinsic pathway in vitro
in vitro leukemic
leukemic cells cells Jurkat,
Jurkat, ML-1
U937, HL-60, K562, U937 MS275MS275 [67] [66]
in vitro leukemic cells Jurkat, U937, HL-60, K562, U937
intrinsic pathway MS275 [67]
expressing Bcl-2, Bcl-XL , CrmA, C8DN, p21 antisense
in vitro prostate cancer LNCaP, bladder cancer T24, breast
intrinsic pathway SAHA [68]
cancer MCF7
in vitro neuroblastoma UKF-NB-4, normoxic and hypoxic
intrinsic pathway VPA [71]
condition
Int. J. Mol. Sci. 2017, 18, 1414 11 of 25
Table 2. Cont.
Nicotinamide,
in vitro 293T, mice embryonal fibroblasts wild t. and
FOXO3 BML-210, [74]
SIRT−/− , Rat1 fibroblasts inducible expressing FOXO3
splitomicin, TSA
in vitro rat cardiomyocytes, in vivo model- a-MHC Beclin
Atg5 & Beclin1 TSA [75]
transgenic mice
siRNA HDAC1,
unknown in vitro cervical cancer HeLa [76]
FK228, SAHA
Atg4D in vitro neuroblastoma BE(2)-C, Kelly, IMR32 siRNA HDAC10 [77]
in vitro cervical cancer HeLa, colon cancer HCT116- SIRT1
Atg5, 7 & 8 - [78]
transfected, starvation
in vitro colon cancer HCT116 Tp53−/−
and +/+ , mouse
FOXO1 TSA [79]
embryonic fibroblasts, liver cancer HepG2
in vitro liver cancer Hep3B, HepG2, Huh7, mouse SAHA, TSA,
Autophagy Akt/mTOR, ULK1 [80]
embryonic fibroblasts MS275
in vitro endometrial stromal sarcoma ESS-1, endometrial
mTOR SAHA [81]
stromal cells HESCs
in vitro uterine sarcoma MES-SA, ESS-1, cervical cancer
p53 HeLa, PANC-1, leukemic cells, pancreatic cancer Jurkat, SAHA [82]
HL-60, U937
in vitro glioblastoma T98G, MEF cells, ULK1/2 knockout
ULK1 SAHA [83]
MEF, ATG3 knockout MEFs
ULK1 in vitro leukemic cells Jurkat SAHA [84]
in vitro PC3, DU145, and HCT116, mouse embryonic
NF- κB SAHA, MS275 [85]
fibroblasts and MEF Atg5−/−
short term culture glioblastoma cells xenografts in
Akt/mTOR SAHA [87]
nude mice
miR-129-5p → GALNT1
in vitro thyroid carcinoma BCPAP, TPC-1, 8505C, CAL62 TSA, SAHA [88]
& SOX4
in vitro breast cancer MDA-MB-231, MCF7, 293T, sodium butyrate,
miR-31→BMI1 [89]
embryonal lung fibroblasts panobiostat
in vitro and mice xenografts leukemic cells/lymphoma RGFP966,
Effect on miR-15 & let-7 → Myc [90]
Daudi, Ramos, Raji, Su-DHL-6, NIH3T3, P493-6 depsipetide
non-coding
RNA Drosha/DGCR8 in vitro 293FT embryonal kidney cells HDAC1 transfected / [91]
miR-449a → HDAC1 in vitro prostate cancer PC-3 siRNA HDAC1 [92]
in vitro liver cancer Huh7, Bel7402, Bel7721, and HepG2,
uc002mbe.2 lncRNA TSA [95]
ShRNA uc002mbe.2 lncRNA
SAHA, VPA,
lncRNA Xist in vitro stem cells from 16 breast cancer cell lines [97]
abexinostat
c-Jun in vitro neuroblastoma SH-SY5Y VPA [98]
ERK in vitro neuroblastoma SH-SY5Y VPA [65]
Effect on signal in vitro colon cancer HT-29 HDAC2 transfected, mice
APC/β-catenin/c-Myc VPA [10]
pathways C57BL/6J-APC-/-
GSK3 β in vitro hematopoetic stem cells VPA [104]
ubiquitin–proteasome in vitro embryonal kidney cells HEK293T VPA, TSA [105]
in vitro embryonic stem cells HDAC1 mutated and wild
Gja1, Irf1, Gbp2 / [106]
type
ACNA2D2 HDAC1 or HDAC2 mutated C57BL/6 mice / [107]
PPAR, ERR HDAC3 conditional C57BL/6 mice / [108]
Anti-angiogenic eNOS, Akt in vitro endothelial cells, Akt transfected, mutatnteNOS / [110]
effect eNOS in vitro HUVEC TSA, MS275 [112]
MMP-2/VEGF in vitro melanoma A375, toxicity tests mice Compound 8 [119]
semaphoring III in vitro HUVEC TSA, SAHA [113]
thrombospondin-1,
in vitro, neuroblastoma BE(2)-C VPA [114]
activin
Int. J. Mol. Sci. 2017, 18, 1414 12 of 25
Table 2. Cont.
in vitro+mice xenografts mouse lung TC-1, rat pancreas

TAP1 & 2, LMP-2,
D11, mouse fibroblasts A9, endothelial cells PA, mouse TSA [118]
tapasin
colinic cells LMD, mouse melanoma B16F10, B16F10/TAP-1
mice C57BL and B6.CB17-Prkdc, human melanoma samples, MGCD0103,
Modulation of MHC class I [120]
mouse melanoma B16-F10-luc LBH589, TSA
immune
response BALB/c and C57BL/6 mice, TCR transgenic mice, HDAC6
STAT3/IL-10 / [121]
recombinant mutants
Tumor associated
in vitro prostate cancer LNCaP, breast cancer MDA-MB-231 SAHA, entinostat [123]
antigens
MHC genes in vitro prostate cancer PCa and DU-145 VPA [126]
5. Combination of HDAC Inhibitors with Other Anticancer Therapeutic

Modalities—Preclinical Studies
The results found in the in vitro and in vivo experiments using various cancer cells have
demonstrated that combination of HDAC inhibitors with anticancer drugs and/or radiotherapy
have synergistic or additive effects [37,140] (see Table 3). Therefore, combinations chemotherapy
and/or radiotherapy with HDAC inhibitors have also been used in clinical trials [141].
Table 3. Combination of HDAC inhibitors with other anticancer therapy.
HDAC
Other Anticancer Therapy Model Effect Ref.
Inhibitor
in vitro prostate cancer LNCaP, breast
Potentiation in ca cells not
SAHA, tubacin etoposide, doxorubicin cancer MCF-7, normal human foreskin [140]
in fibroblasts
fibroblast cells
TSA 5-aza-20 -deoxycytidine in vitro pancreatic endocrine cancer cells Potentiation [52]
in vitro LNCaP, DU-145, and PC-3
TSA 5-aza-20 -deoxycytidine Potentiation [55]
prostate cancer
SAHA, TSA 5-aza-20 -deoxycytidine in vitro ovarian cancer Hey, SKOv3 Synergism [142]
VPA 5-aza-20 -deoxycytidine in vitro leukemic cells HL-60, MOLT4 Synergism [143]
VPA 5-aza-20 -deoxycytidine Ptch knockout mice Potentiation [144]
in vitro Kasumi-1 cells and blasts from
Depsipeptide 5-aza-20 -deoxycytidine Potentiation [145]
patient with t(8;21) AML
TSA 5-aza-20 -deoxycytidine in vitro lung cancer A549, H719 Synergism [146]
in vitro leukemia cells HL60, HL60/LR,
SAHA β-phenylethyl isothiocyanate HL60/C6F, U937, ML1, samples from Potentiation [147]
patients with acute myeloid leukemia
in vitro lung cancer HCC827, A549,
Panobinostat erlotinib Synergism/Potentiation [148]
NCI-H460 (EGFR wild type and mutant)
in vitro and BALB/cAJcl-nu/nu mice
3-deazaneplanocin A (EZH2
SAHA xenografts lung cancer NCI-H1299, Synergism [149]
inhibitor)
NCI-H1975, A549, PC-3
sodium
in vitro multiple myeloma U266, RPMI8226
butyrate, bortezomib Synergism [150]
and patient samples
SAHA
in vitro and nude mice xenografts,
SAHA bortezomib pancreatic cancer L3.6pl, pancreatic Potentiation [151]
epitelium HPDE6-E6E7
lymphoma cell SUDHL16, SUDHL4 ,
SUDHL6, OCI-LY10, OCI-LY3
SAHA carfilzomib Potentiation [152]
bortezomib-resistant SUDHL16-10BR,
OCI-LY10-40BR and lymphoma cells
frompatients
SNDX-275,
carfilzomib, bortezomib lymphoma cell Granta 519, Rec-1, HF-4B, Potentiation [153]
SAHA
JVM-2, MINO, JVM-13
Int. J. Mol. Sci. 2017, 18, 1414 13 of 25
Table 3. Cont.
HDAC
Other Anticancer Therapy Model Effect Ref.
Inhibitor
Potentiation-schedule
VPA cisplatin, etoposide in vitro neuroblastoma cell UKF-NB-4 [154]
dependent
belinostat, in vitro lung cancer H82, H146, H526 Synergism-schedule
cisplatin, etoposide [155]
romidepsin and H446 dependent
Potentiation-schedule
VP-16, ellipticine,
in vitro glioblastoma U118, brest cancer dependent, except of
5fluorouracil, doxorubicin,
TSA, SAHA MCF-7, normal breast MCF-12F, normal 5fluorouracil, [156]
cisplatin, cyclophosphamide,
intestinal epithelia FHs74Int cyclophosphamide,
campthotecin
campthotecin
in vitro anaplastic thyroid carcinoma
VPA paclitaxel Potentiation [157]
CAL-62, ARO
VPA Gene therapy (MSC + HSV-TK) glioblastoma U87 xenografts in nude mice Potentiation [158]
MSC + HSV-TK- herpes simplex thymidine kinase transfected bone marrow mesenchymal stem cells.
HDAC inhibitors were combined with other epigenetic modifiers. Inhibitors of DNA methyl
transferases azacitidine and decitabine had increased antitumor effects on myelodysplastic
syndrome, prostate, ovarian and pancreatic endocrine tumors cells when used with HDAC
inhibitors [52,55,142,159,160]. Decitabine and VPA both induced apoptosis and the combination
increased their effects both in vitro and in vivo on leukemic cells and on medulloblastoma and
rhabdomyosarcoma that develop in Ptch1 (Protein patched homolog 1) knockout mice [143,144].
On the contrary, this combination induced the CD133 expression in neuroblastoma cells with the
methylated CD133 promotor [133]. Co-treatment of several cancer cells (prostate, pancreatic, lung
and AML) with TSA and decitabine synergistically induced apoptosis [52,55,145,146]. In addition,
tranylcypromine (monoamine oxidase inhibitor) and SAHA showed synergistic enhancement of
apoptosis in glioblastoma cells [58].
Positive effects have been reported for combinations of HDAC inhibitors and ROS-generating
agents. Adaphostin (a tyrosine kinase inhibitor that induces intracellular ROS) potentiates entinostat
and SAHA induced apoptosis in leukemia cells and depletion of ROS scavenger GSH potentiates the
anti-leukemic effect of SAHA [58,147]. Panobiostat sensitized lung adenocarcinoma cells including
cells with K-ras (Kirsten rat sarcoma viral oncogene homolog) or epidermal growth factor receptor
(EGFR) mutation to the anti-proliferative effects of the tyrosine kinase inhibitor erlotinib in the in vitro
experiment [148]. EZH2 (catalytic subunit of polycomb repressive complex 2) interacts with class
I HDACs and transcriptional repression by EZH2 requires the activity of the HDACs and HDAC
downregulates PRC2 proteins. Therefore, one may speculate that concurrent inhibition of these
epigenetic silencing enzymes has synergistic antitumor effects. Co-treatment non-small cell lung cancer
(NSCLC) cells with 3-deazaneplanocin A (EZH inhibitor) and vorinostat synergistically induced apoptosis
in all tested NSCLC cell lines, independently on their EGFR status. The co-treatment by EZH and HDAC
inhibitors induced accumulation of p27Kip (Cyclin dependent kinase inhibitor p27) and a decrease in
cyclin A, suppressed EGFR signaling, both in EGFR-wild-type and mutant NSCLC cells [149].
Another effective combination of HDAC inhibitors is that with proteasome inhibitors. Cancer cell
death due to a combination of proteasome and HDAC inhibitors is caused by induction of oxidative
stress, endoplasmic reticulum stress and stimulations of JNK (Jun-N-terminal kinase). Treatment of
multiple myeloma cells with bortezomib made the cells more sensitive to HDAC inhibitors [150].
Trials in patients with multiple myeloma demonstrated an increase in SAHA antitumor effects in
combination with bortezomib [161,162]. Proteasome inhibitor marizomib potentiates apoptosis induced
by SAHA or entinostat in pancreatic cancer cells [151]. The in vitro and in vivo studies with lymphomas
cells including bortezomib resistant ones showed that proteasome inhibitor carfilzomib increased the
anticancer effect of SAHA [152,153].
Numerous studies show synergisms or additive effects of HDAC inhibitors and DNA damaging
agents such as topoisomerase inhibitors, DNA intercalators, inhibitors of DNA synthesis, covalently
Int. J. Mol. Sci. 2017, 18, 1414 14 of 25
modifying DNA agents (i.e., doxorubicin, epirubicin, etoposid, CDDP, melphalan, and temozolomide)
and ionizing radiation in many cancer cell lines (reviewed in [37]). VPA combined with CDDP
or etoposide (VP-16), but not with vincristine, was found to act synergistically. These results
indicate that HDAC inhibitors increase the cytotoxic efficiency of the only of several DNA-damaging
anticancer drugs. The mechanisms of the potentiating effects of HDAC inhibitors have not yet been
fully explained. The sequence of drug application is important for sensitizing neuroblastoma cells
to CDDP and VP-16 by VPA. It potentiates the cytotoxic effect of CDDP or VP-16 only when added
simultaneously, or when cells were preincubated with cytostatics before exposure to HDAC inhibitors.
In contrast, the reversed sequence (pretreatment of cells with VPA) did not give any further increase
in cytotoxicity of cytostatics [154]. The results found by Luchenko et al. [155] indicated that DNA
relaxation is not required for the synergy of belinostat and romidepsin with CDDP and VP-16 in
small cell lung cancer cells. One can speculate that the changes in the structure of DNA caused by
CDDP and VP-16 (formation of DNA adducts or DNA cross-links by CDDP, intercalation of VP-16
into DNA, and formation of ROS by both drugs) increase accessibility of nucleosomal core histones to
their acetylation, which additionally determines transcription of some genes involved in DNA repair
or apoptosis. Kim et al. described that pre-treatment of cells (glioblastoma lines D54 and U118, breast
cancer MCF-7, normal breast MCF-12F and intestinal FHs74Int) with SAHA or TSA enhances the
cytotoxicity of VP-16, ellipticine, doxorubicin, and CDDP but not that of drugs which do not target
the DNA, such as 5-fluorouracil [156]. It only partially agreed with our results, Kim et al. [156] found
potentiation of cytotoxicity after pretreatment of cells with HDAC inhibitors before exposure to other
drugs, while the results of our experiments indicated the opposite phenomenon; the administration of
VPA after DNA-damaging cytostatic increased their cytotoxicity [154]. Nevertheless, both groups used
different HDAC inhibitors and different cell lines.
HDAC inhibitors are able to deacetylate α-tubulin which has stabilizing effect on microtubules.
There was found that combination of TSA and paclitaxel increases apoptosis induction in endometrial
and anaplastic thyroid carcinoma cells [138,157].
The in vitro experiments and the experiments with mice in vivo showed that the combination
of VPA and temozolomide enhanced the apoptotic and autophagic cell death, as well as
suppressed the migratory activities in temozolomide-resistant glioblastoma cells that express
O-(6)-methylguanine-DNA methyltransferase (MGMT). The effect of VPA is caused by downregulation
of MGMT [158].
6. Clinical Studies and Registered Drugs

As mentioned above, several HDAC inhibitors are tested in clinical studies and four of them are
approved drugs (vorinostat, belinostat, panobinostat and romidepsin). The most common toxicities
of those HDAC inhibitors, which are not the class-specific and have been observed in all their types,
are thrombocytopenia, neutropenia, diarrhea, nausea, vomiting and fatigue [163].
SAHA had only the modest activity as a single agent (response rate is 10–20% in AML and
myelodysplastic syndrome (MDS)). However, a combination with 5-azacitidine increased its response
rate by 30%. The combination of SAHA with idarubicin and cytarabine had synergistic activity
that was maximal when SAHA preceded cytarabine and there were responses in all patients with
AML with FLT3/ITD mutation, which is otherwise associated with a worse prognosis [164,165].
Administration of SAHA in refractory CTCL patients showed in phase II trials objective response
in nearly 30% [166,167]. HDAC inhibitor silences the genes in common translocations associated
with hematological malignancies, e.g., those of the AML/ETO fusion protein [168]. Phase I study of
patients with advanced leukemia and MDS treated with SAHA showed clinical benefit in AML and
MDS [169]. The clinical phase II study proved that panobinostat is effective in relapsed/refractory
Waldenstrom macroglobulinemia [170]. Mocetinostat had the only limited efficacy as showed the
clinical study phase II for the treatment of patients with refractory chronic lymphocytic leukemia
(CLL). Therefore, the combination with other agents such as conventional chemotherapeutic drugs
Int. J. Mol. Sci. 2017, 18, 1414 15 of 25
was recommended [171]. Panobinostat underwent phase I and II clinical studies for the treatment of
both solid and hematologic malignancies and phase III clinical trials for CTCL and chronic myeloid
leukemia that showed promising results against CTCL [172] and leukemias, respectively, and proved
the increased acetylation of histones in malignant cells that was associated with apoptosis [173].
Panobinostat also underwent phase III clinical trials against CTCL and leukemia in an oral form
and showed the positive effect. Despite the promising results in the treatment of CTCL, SAHA and
romidepsin have not been effective in studies with different solid tumors (neuroendocrine tumors,
glioblastoma multiforme, mesothelioma, refractory breast, colorectal, NSCLC, prostate, head and neck,
renal cell, ovarian, cervical and thyroid cancers) and Hodgkin lymphoma [174,175].
Therefore, the clinical trials tend to combine HDAC inhibitors with other drugs to enhance
their anticancer effects. A clinical study showed that VPA increased efficacy of radiochemotherapy
with temozolomide in glioblastoma patients [176]. VPA with doxorubicin appeared to have the 16%
response rate in patients suffering from refractory or recurrent mesothelioma [177]. SAHA improved
effect of carboplatin and paclitaxel in NSCLC [178] and reversed resistance to tamoxifen in estrogen
receptor positive breast cancer [179]. Phase II study with a combination of erlotinib with entinostat
showed prolonged progression-free survival in NSCLC patients harboring high E-cadherin levels,
irrespective of the EGFR genotype compared to erlotinib monotherapy, while does not improve the
outcome of patients with low E-cadherin [180]. Clinical trial with the combination of the immune
checkpoint therapy with the HDAC inhibitor is ongoing [181].
7. Conclusions and Future Direction

HDACs are involved in different cellular pathways and functions; nevertheless, further studies are
necessary to disclose all their functions and cellular interactions, which might result in development
of more efficient therapy with HDAC inhibitors. HDAC inhibitors seem to be a promising group
of anti-cancer drugs, particularly in combination with other anti-cancer drugs and/or radiotherapy.
Of HDAC inhibitors, SAHA and romidepsin have been approved for CTCL, romidepsin also for PTCL,
belinostat for therapy of PTCL and panobinostat for multiple myeloma. Many other HDAC inhibitors
are in clinical trials for the treatment of both hematological and solid malignancies. Even though many
biological effects of HDAC inhibitors have been found, explanations of these effects remain unclear.
In addition, their use in the combination with other drugs and the schedule of such drug
combinations need to be investigated, in both preclinical and clinical studies. Indeed, recently, we have
found that sequence of HDAC inhibitor and DNA-damaging drug is important for increase of
cytotoxicity [154]. The discovery of predictive factors for evaluation of HDAC inhibitors is also
necessary as demonstrated in the study with combination of erlotinib with entinostat [180]. The other
most important question is whether the pan-HDAC inhibitors or the selective inhibitors will be more
efficient in different types of cancers. Furthermore, assumption about the role of some HDAC inhibitors
as inducers of cancer stem cells [133,134] or about the phenomenon that HDAC inhibition may enhance
the epithelial–mesenchymal transition of cancer cells [129,130] needs further studies. We may conclude
that while the preliminary results are promising, large multicentric clinical studies are needed to
ascertain whether this treatment offers beneficial clinical outcomes with tolerable side-effect profiles.
However, the great potential for epigenetic therapies that is caused by the fact that epigenetic
changes are reversible might be considered.
Acknowledgments: This work was supported by grant GA CR 17-12816S and by the Ministry of Health of the
Czech Republic for conceptual development of research organization 00064203 (University Hospital Motol, Prague,
Czech Republic).
Author Contributions: Tomas Eckschlager and Jan Hrabeta wrote the manuscript; Johana Plch contributed to the
figure and table design and the literature research; and Marie Stiborova revised and approved the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Int. J. Mol. Sci. 2017, 18, 1414 16 of 25
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International Journal of

Received: 14 May 2017; Accepted: 27 June 2017; Published: 1 July 2017

Keywords: histone deacetylases; histone deacetylase inhibitors; cancer; apoptosis; autophagy;

Int. J. Mol. Sci. 2017, 18, 1414; doi:10.3390/ijms18071414 www.mdpi.com/journal/ijms

2. Acetylation and Deacetylation

3. HDAC Inhibition and Its Effects

Table 1. Overview of selected histone deacetylases (HDAC) inhibitors.

Class HDAC Inhibitor Target HDAC Class Clinical Status

4. Mechanisms of HDAC Inhibitors Action

4.1. Cell Cycle Arrest

4.2. Apoptosis Induction

4.3. The Effects on Induction of Autophagy

4.4. The Effects on Non-Coding RNA

4.5. The Effects on Cellular Signaling Pathways

4.6. The Antiangiogenic Effect

4.7. HDAC Inhibitor-Induced Modulation of Immune Response

4.8. The Effects on Stem Cells

4.9. Other Effects of HDAC Inhibitors

p53 in vitro lung cancer A549 depsipeptide [47]

in vitro+mice xenografts mouse lung TC-1, rat pancreas

5. Combination of HDAC Inhibitors with Other Anticancer Therapeutic

Table 3. Combination of HDAC inhibitors with other anticancer therapy.

6. Clinical Studies and Registered Drugs

7. Conclusions and Future Direction

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