Send Orders for Reprints to reprints@benthamscience.

net
Current Molecular Pharmacology, 2020, 13, 1-0 1
RESEARCH ARTICLE

Epigenetic Evaluation of N-(2-hydroxyphenyl)-2-Propylpentanamide, a

Valproic Acid Aryl Derivative with Activity Against HeLa Cells
GR Luna-Palencia1, J Correa-Basurto2, J Trujillo-Ferrara3, MA Meraz-Ríos4 and
I Vásquez-Moctezuma5,*

1
Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Pol-
itécnico Nacional, Av. IPN 2508, San Pedro Zacatenco, CDMX, 07360, México; 2Laboratorio de Diseño y Desarrollo
de Nuevos Fármacos e Innovación Biotecnológica (Laboratory for the Design and Development of New Drugs and
Biotechnological Innovation), Escuela Superior de Medicina, Instituto Politécnico Nacional, Plan de San Luis y Díaz
Mirón s/n, Casco de Santo Tomás, CDMX, 11340, México; 3Laboratorio de Bioquímica, Laboratorio de Modelado
Molecular, Bioinformática y Diseño de Fármacos de la Escuela Superior de Medicina, Instituto Politécnico Nacional,
Plan de San Luis y Díaz Mirón s/n, Casco de Santo Tomás, CDMX, 11340, México; 4Departamento de Biomedicina
Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; 5Laboratorio de Mor-
fología de la Maestría en Ciencias de la Salud, Escuela Superior de Medicina, Instituto Politécnico Nacional, Plan de
San Luis y Díaz Mirón s/n, Casco de Santo Tomás, CDMX, 11340, México

Abstract: Background: Valproic acid (VPA) is an HDAC inhibitor (HDACI) with an anticancer
activity, but it is hepatotoxic. N-(2-hydroxyphenyl)-2-propylpentanamide (o-OH-VPA) is a VPA
aryl derivative designed in silico as a selective inhibitor of HDAC8 with biological properties
against HeLa, rhabdomyosarcoma and breast cancer cell cultures.

Objective: Objective: We studied the epigenetic mechanism of o-OH-VPA as an HDACI and we

evaluated whether it was toxic to normal cells.

ARTICLE HISTORY
Received: February 06, 2020
Revised: May 14, 2020
Accepted: May 25, 2020
DOI:
10.2174/1874467213666200730113828
Methods: HeLa cells and primary human fibroblasts were used for this study as carcinogenic and
normal cells, respectively. Cell survival was evaluated by MTT assay, whereas viability and dou-
bling time were determined by the Trypan-blue method. HDAC activity was tested using the colori-
metric HDAC activity assay. The expression of p21 was analyzed by PCR and HDAC8 expression
was also evaluated by real-time PCR. Cell cycle and caspase-3 activity were analyzed by flow cy-
tometry and caspase-3 colorimetric assay, respectively.
Results: o-OH-VPA (IC50 = 0.1 mM) was fifty-eight times more effective than VPA (IC50 = 5.8
mM) to reduce HeLa cell survival. Furthermore, o-OH-VPA increased the doubling time of HeLa
cells by 33% with respect to the control. o-OH-VPA acted as HDACI in HeLa cells without affect-
ing the HDAC8 expression, arresting the cell cycle of HeLa cells in the G0/G1 phase due to the in-
crease in p21 expression with the inhibition of caspase-3 activity without exhibiting toxicity to-
ward normal cells.

Conclusion: Our results revealed that o-OH-VPA is an HDACI with a selective effect against
HeLa cells but without the known toxicity exerted by most pan-HDACIs on normal cells.

Keywords: Anticancer agents, histone deacetylase 8 inhibitor, valproic acid, aryl valproic acid derivative, HeLa cells, epigenet-
ic drugs.

1. INTRODUCTION E-mail: g17isma65@gmail.com

Reversible acetylation of histones is an epigenetic modi-
fication which is involved in the regulation of gene expres-
sion that is carried out by acetyltransferase and histone
deacetylase (HDACs) enzymes. These modifications con-
trol the in-
* Address correspondence to this author at the Morfolog, Escuela Superior
de Medicina del IPN, Salvador D, Mexico; Tel: 5557296000;
1874-4672/20 $65.00+.00
teraction of positively charged histones with negatively
charged DNA, and this regulates chromatin conformation
and transcriptional activity [1]. This control occurs because
HDACs remove the acetyl group of lysine residues located
at the N-terminal tails of the histones, leading to a more
closed chromatin conformation that hinders the accessibility
of transcription factors to DNA [2]. In addition, HDACs con-
trol the acetylation status and function of numerous non-his-
© 2020 Bentham Science Publishers
2 Current Molecular Pharmacology, 2020, Vol. 13, No. 0
tone proteins and transcription factors [3]. Since epigenetic
alterations have been associated with cancer, new treatments
are being under developement based on inhibitors of histone
deacetylation [4-6]. HDACs are involved in regulating pro-
cesses such as the cell cycle, differentiation, apoptosis, mi-
gration, invasion and angiogenesis of cancer cells [7]. At
this time, there are 18 HDAC family members that are
grouped into four classes: class I (1, 2, 3 and 8); class II,
which is further divided into classes IIa (4, 5, 7 and 9) and
IIb (6 and 10); class III (12-18) and class IV (11). Classes I,
II, and IV are also called “classical” HDACs, and all these
have Zn 2+ cofactor, whereas class III includes HDACs that
are nicotinamide adenine dinucleotide (NAD+) dependent, re-
ferred to as sirtuins [8, 9]. Since there are differences in the
activity of histone deacetylases inhibitors (HDACIs) against
specific classes of HDACs, these may contribute to their tu-
mor cell selectivity observed. The fatty acid valproic acid in-
hibits class I HDACs 1, 2, 3 and 8 in the mM scale, but it is
ineffective against HDACs 6, 7 and 9 [10]; The very high
concentration required for its anti-tumor activity is one of
the major drawbacks [7], for instance, sodium valproate in-
duces mitochondrial respiration dysfunction in HepG2 cells
at 2 mM [11].
HDAC8 is a class I HDAC related to carcinogenesis and
cancer progression, and its expression levels are elevated ap-
proximately 10-fold in HL-60, HeLa S3, K-562, MOLT-4,
SW480, A549 and G361 cancer cell lines compared to nor-
mal tissues [12]. Moreover, HDAC8 is associated with the
regulation of the proliferation, clonogenic growth, and neuro-
nal differentiation of neuroblastoma cells and its high expres-
sion correlates with neuroblastoma progression and poor sur-
vival prognoses [13]. HDAC 8 can be targeted using com-
pounds that have a chemical scaffold with metal chelators or
hydrophobic properties. Our research group has been fo-
cused on drug design for some of the ligands with hydropho-
bic moieties (from valproic acid) and amino acids [14, 15]
or aryl derivatives [16, 17]. N-(2-hydrox-
yphenyl)-2-propylpentamide (o-OH-VPA) is a valproic acid
(VPA) aryl derivative (Fig. 1) designed in silico by mixing
VPA with the arylamine core of suberoylanilide hydroxamic
acid (SAHA) that shows biological properties against human
cancer [16, 17]. However, the pharmacological mechanism
of o-OH-VPA remains unknown.
This work aimed to evaluate the effect of o-OH-VPA on
the proliferation of HeLa cells by MTT and trypan blue as-
says, as well as its action as HDACI by determining the total
enzymatic activity of HDACs, expression of HDAC8, analy-
sis of the cell cycle and caspase-3 activity. Moreover, we al-
so tested the toxicity to normal cells (primary human fi-
broblasts).
2. MATERIALS AND METHODS
2.1. Cell Cultures
HeLa cell line derived from cervical cancer and human
primary dermal fibroblast ATCC PCS-201-012 (FB) were
Luna-Palencia et al.
cultured in Dulbecco-Modified Eagle’s Medium (DMEM
Glutamax, Gibco, Life-technologies, Invitrogen, USA) sup-
plemented with 10% (v/v) heat-inactivated fetal bovine
serum (FBS, Biowest, Kansas City, MO, USA) and an antibi-
otic-antimycotic reagent (Gibco). The cell cultures, both un-
treated and treated with VPA or o-OH-VPA, were main-
tained at 37°C in a humidified atmosphere containing 5%
CO2. DMSO was used as a drug vehicle in all the cell cul-
ture experiments.
2.2. MTT Assay
3
HeLa cells (1 x 10 cells per well) or human fibroblast (F-
3
B) cells (3 x 10 cells per well) were seeded in 96-well cul-
tured plates in 100 µL DMEM containing 10% heat-inacti-
vated FBS. After 24 h, HeLa cells were treated with fresh
medium containing VPA (2, 4, 6 and 8 mM) or o-OH-VPA
(0.05, 0.1, 0.2, 0.4 and 0.5 mM); On the other hand, FB cells
were treated with fresh medium containing 5 mM VPA or
0.1 mM o-OH-VPA (IC50 for HeLa cells), so as to evaluate
their cytotoxicity in non-tumor cells. The plates were incu-
bated for 48 h and were analyzed for cell survival using the
colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetra-
zolium bromide (MTT) dye reduction assay (Sigma-Aldrich,
St. Louis, MO, USA) [14].
2.3. Trypan Blue Exclusion Method
HeLa cells (10 x 103 cells per well) were seeded in 24-
well cultured plates in 500 µL DMEM containing 10% FBS.
After 24 h, HeLa cells were treated with fresh medium con-
taining VPA (2, 4, 6, 8 and 10 mM) or o-OH-VPA (0.1, 0.2,
0.3, 0.4 and 0.5 mM). Plates with different concentrations of
the compounds were incubated for 48 h, and those with the
same concentration were incubated for 24, 48 and 72 h. The
viability, or live cells per well, was determined by the Try-
pan-blue exclusion method. For this, adherent and floating
cells were collected, and 0.4% Trypan-blue solution (Sig-
ma-Aldrich, St. Louis, MO, USA) was added 1:1 (v/v) and
counted in a TC10 Automated Cell Counter (BIO-RAD, Lab-
oratories, CA, USA). The doubling time was calculated with
0, 24 and 48 h data [18].
2.4. HDAC Activity Assay
Assays for HDAC activity were performed using the col-
orimetric HDAC activity assay (BioVision Research Prod-
ucts, Mountain View, CA, USA), which eventually detects
the activity of all HDACs that can deacetylate histone. HeLa
nuclear protein (50 µg) was obtained as described in a previ-
ous study [14] and was diluted with 83 µL of ddH2O and 2
µL of both 250 mM VPA or 5 mM o-OH-VPA as appropri-
ate, followed by 10 µL of the 10X HDAC assay buffer, and
then 5 µL of the HDAC substrate. After incubating the plate
for 1 h at 37°C, the reaction was ended with 10 µL of Lysine
Developer, subsequently, the solution was incubated for 30
min at 37°C. Finally, the absorbance at 405 nm was read us-
ing a microplate reader.
Epigenetic Evaluation of N-(2-hydroxyphenyl)-2-Propylpentanamide
2.5. Total RNA Extraction, Reverse Transcription and
Polymerase Chain Reaction to Evaluate the Expression
of HDAC8 and p21
Total RNA was isolated with TRIzol (Invitrogen CA,
USA) according to the manufacturer’s instructions. RNA in-
tegrity was electrophoretically verified with 1% agarose gel
by ethidium bromide staining, and RNA purity was verified
by an OD260/OD280 nm absorption ratio >1.8. The total RNA
(0.5 µg) of HeLa cells, untreated or treated with 5 mM VPA
or 0.1 mM o-OH-VPA for 24 h, was reverse transcribed
with the Maxima First Strand cDNA Synthesis kit (Thermos-
cientific Pittsburgh, USA), and a polymerase chain reaction
was conducted using the PCR Master Mix (2X) (Thermosci-
entific Pittsburgh, USA). Primer design was performed us-
ing the Oligo Explorer software 1.1.2, hence the primer se-
quences are shown in Table 1, including GAPDH primers as
housekeeping gene control. The thermal cycling conditions
for each primer set were 95°C for 3 min followed by 35 cy-
cles which are as follows: denaturation step at 95°C for 30 s;
annealing at 50°C for 30 s; and extension at 72°C for 30 s.
Finally, a final extension cycle was run at 72°C for 5 min.
PCR products were visualized on a 3% agarose gel by ethidi-
um bromide staining.
2.6. Real-Time Quantitative PCR
Real-time qPCR was performed using the Power SY-
BR® Green PCR Master Mix (2X), purchased from Applied
Biosystems. Reactions (10 µL) were conducted using a Ste-
pOne Real-Time PCR System, in which the concentrations
of primers were each 150 nM and 1 µL of cDNA diluted to
1/10. The thermal cycling conditions for each primer set
were 95°C for 10 min followed by 40 cycles as follows: de-
naturation step at 95°C for 15 s and annealing at 60°C for 60
s. Finally, the melt curve was obtained. The experiment was
carried out in triplicate, and each sample was also run in tri-
plicate. The relative expression to GAPDH was determined
using the Pfaffl method [19].
2.7. Cell Cycle and Apoptosis Analysis
Unsynchronized HeLa cell cultures were treated with 5
mM VPA or 0.1 mM o-OH-VPA for 48 h. Adherent and
floating cells were collected by centrifugation and were
fixed in cold 70% ethanol. Fixed cells were washed twice
with cold PBS and treated with 0.2 mg/mL RNAse A and lat-
er stained with 40 µg/mL propidium iodide. The samples
were analyzed using an LSR Fortessa flow cytometer (BD
Biosciences). Cell cycle and apoptosis analysis were deter-
mined by the ModFit LT program.
2.8. Caspase-3 Assay
Assays were performed using the Caspase-3/CPP32 col-
orimetric assay kit (BioVision Research Products, Mountain
View, CA, USA) according to the instructions by the manu-
factures. Cytosolic protein (100 µg) of HeLa cells treated
with 0.5% DMSO (D, vehicle), 5 mM VPA or 0.1 mM o-O-
Current Molecular Pharmacology, 2020, Vol. 13, No. 0 3
H-VPA were diluted with 50 µL cell lysis buffer and 50 µL
of 2X Reaction Buffer (containing 10 mM DTT). DEVD-p-
NA substrate was added to achieve a final concentration of
200 µM, and this mixture incubated at 37°C for 2 h. In addi-
tion, for each sample, a blank was prepared equal to its corre-
sponding sample, but without adding the substrate, for sub-
tracting background. Samples were read at 405 nm in a mi-
crotiter plate reader.
2.9. Statistical Analysis
Data were expressed as the mean ± SD. One or two-way
ANOVA followed by Dunnett’s multiple comparisons test
was performed, as would be appropriate, using GraphPad
Prism version 6.00 for Windows, GraphPad Software, La Jol-
la California USA (www.graphpad.com).
3. RESULTS
The concentration-response curve (Fig. 2A) showed that
either o-OH-VPA or VPA reduced the HeLa cell survival in
a concentration-dependent manner, but o-OH-VPA was fif-
ty-eight times more effective than VPA (0.1 mM vs 5.8
mM). Moreover, the membrane integrity, and consequently
the viability of the HeLa cells, was reduced to 80.3 ± 8.6%
by o-OH-VPA compared to control cells near the IC50 ob-
tained by MTT at 48 h (Fig. 2B).
The number of living HeLa cells per well, evaluated by
the trypan blue assay decreased 48% after 48 h of treatment
with 0.1 mM o-OH-VPA, was similar to the cells treated
with 5 mM VPA, compared with 0.5% DMSO (vehicle);
that is, from 89 X 103 to 43 X 103 cells. At 72 h of treatment
with o-OH-VPA treatment, the number of living HeLa cells
per well decreased by 75% and 51% with respect to the con-
trol and VPA. The doubling time of HeLa cells treated with
0.1 mM o-OH-VPA (15.4 h) was 33% and 7% higher than
that observed in HeLa cells treated with 0.5% DMSO (11.6
h) or 5 mM VPA (14.5 h), respectively (Fig. 3).
The total activity of HDACs in HeLa nuclear protein ex-
tract treated with 0.1 mM o-VPA-OH was reduced only by
23% compared to approximately 90% caused by 5 mM VPA
treatment (Fig. 4). In contrast, the overexpression of
HDAC8 observed in the HeLa cells treated with 5 mM VPA
was not observed in the cells treated with 0.1 mM o-OH-V-
PA (Fig. 5).
The decrease in the number of viable HeLa cells caused
by 0.1 mM o-OH-VPA was associated with the relative in-
crease in p21 expression (Fig. 6A), as well as an increase
(7.4%) in the numbers of HeLa cells in the G0/G1 phase
(58.6 ± 1.4%) compared to the cells treated with DMSO
(51.2 ± 1.2%), and the decrease in the cell cycle S phase
(Fig. 6B). Finally, it was observed that the activity of cas-
pase-3 (Fig. 6C), as well as apoptosis induction (Fig. 6B) in
HeLa cells treated with 0.1 mM o-OH-VPA, were reduced
by almost 50% with respect to 0.5% DMSO-treated HeLa
cells.
Despite its toxic effect on HeLa cells (cancer cells), o-O-
H-VPA did not show toxicity towards normal cells, such as
4 Current Molecular Pharmacology, 2020, Vol. 13, No. 0
primary cultures of human fibroblasts, when they were chal-
lenged with the IC50 obtained in HeLa cells. In contrast,
VPA 5 mM reduced the fibroblast cell survival to 66.5 ±
3.7% (Fig. 7), demonstrating a selective toxic effect of o-O-
H-VPA toward HeLa cancer cells.
4. DISCUSSION
Most currently used HDACIs act rather non-selectively
and inhibit either all or at least several members of the
HDAC family [7]. Our research group is focused on the ra-
tional design of selective HDACIs by using particularly crys-
tal structures of HDAC8 as non-HDAC selective inhibitors
approved by the FDA caused a variety of side-effects such
as bone marrow depression, diarrhea, weight loss, taste dis-
turbances, electrolyte changes, disordered clotting, fatigue,
and cardiac arrhythmias [7].
Our results showed that o-OH-VPA had anti-prolifera-
tive properties against HeLa cells and was fifty-eight times
more effective than VPA, as demonstrated by its IC50 that
was determined using the MTT assay (Fig. 2). The o-OH-V-
PA affected the membrane integrity more than VPA, decreas-
ing the viability of HeLa cells as a cytotoxic effect in a con-
centration-dependent manner. Furthermore, 0.1 mM o-O-
H-VPA decreased the proliferation of HeLa cells by increas-
ing the doubling time in more than 30% compared to the
cells that were treated with 0.5% DMSO as the control, with-
out affecting the viability after 48 h of treatment. o-OH-V-
PA was designed in silico as a selective inhibitor of HDAC8
[17], and it has a similar effect as an HDACI in HeLa cells;
o-OH-VPA was recently reported to have an affect on HMG-
B1, an essential enzyme with high expression in tumor cells
which is dependent on its acetylation state [20]. VPA inhibit-
ed approximately 90% of HDACs at 5 mM treatment,
whereas for o-OH-VPA at 0.1 mM, it was only 20%. In con-
trast to VPA, which has been reported to be a class I
HDACs (HDAC1, 2, 3, and 8) inhibitor that induces the
acetylation of histone H4 and slightly inhibits the acetyla-
tion of histone H3 [10], o-OH-VPA could be a specific
HDAC8 inhibitor. HDAC8 is primarily localized in the nu-
cleus [21] but appeared to be more concentrated in the sub-
nuclear regions rather than be distributed evenly throughout
the entire nucleus. This observation suggests that HDAC8
may act on other proteins in addition to histones and may
have only limited deacetylation activity on histone proteins;
The activity of HDAC8 is low compared to that of HDAC1,
and its precise target of deacetylation has not yet been identi-
fied [12]. According to recent results, however, o-OH-VPA
could also inhibit other HDACs, according to recent results,
because o-OH-VPA helps HMGB1 migration from the nu-
cleus to the cytoplasm [20]. Waltregny and coworkers found
that HDAC8 is predominantly cytosolic, both in human tis-
sues and in vitro-grown human vascular smooth muscle
cells, where it displays a cytoskeleton-like pattern of expres-
sion and may colocalize with α-actin [22]. Some selective
HDAC8 inhibitors have been developed, such as PCI-34051,
but when compared to other broad-spectrum HDAC inhibi-
tors, they do not cause detectable acetylation of histones or
tubulin [23], This has been consistent with our results of the
Luna-Palencia et al.
total activity of HDACs in HeLa nuclear protein extract treat-
ed with 0.1 mM o-OH-VPA since only a decrease of 23%
was observed.
o-OH-VPA at 0.1 mM did not increase HDAC8 gene ex-
pression, as was the case for the 5 mM VPA, which is inter-
esting because HDAC8 is important for human tumor cell
lines growth since the proliferation of lung, colon and cervi-
cal cancer cell lines is reduced after HDAC8 knockdown
[24], This reduction indicated that the inhibition of HDAC8
is implicated in the decrease of proliferation.
o-OH-VPA induced the overexpression of p21, which is
a potent inhibitor of most cyclin/CDK complexes that result
in an accumulation of cells in the G0/G1 phase, preventing
cells from shifting to the S-phase [25] and inhibiting cas-
pase-dependent apoptosis in HeLa cells. Reduced growth
will protect the cell against long-term genome damage
caused by replicative stress through the S-phase and chromo-
some missegregation during mitosis [26]. Previous studies
have demonstrated that the cell cycle inhibitor p21 is com-
monly induced in response to HDACI [27, 28] and plays a
key role in the G1 cell cycle arrest of many cancer cells
[29]. The progression through the G1-S checkpoint is neces-
sary for the induction of apoptosis by HDACIs, such as SA-
HA, oxamflatin and depsipeptide, because cells arrested in
the G1 phase are resistant to HDACI-induced death [30].
The above was observed with o-OH-VPA, where apoptosis
and caspase-3 activity were decreased as a result of G0/G1
arrest. It is important to determine whether cell cycle arrest
is a permanent state due to DNA damage caused by o-OH-V-
PA because cellular senescence, which is a potent tumor sup-
pressive mechanism, can be activated [31].
o-OH-VPA did not show toxicity for normal cells, as ob-
served with the human fibroblasts used in this work. The
mechanism by which HDACIs exhibit selective toxicity for
cancer cells has not been defined in its entirety, Still there is
a close relationship between histone acetylation and the tran-
scriptional activity of specific genes that are involved in pro-
liferation and/or apoptosis [32]. For example, it has been de-
monstrated that HDACs 8 and 6 are overexpressed in HeLa
cells [33]. In contrast, it had previously been demonstrated
that fibroblasts could suffer extensive damage in the pres-
ence of HDACIs [34]. HDACIs cause rapid and extensive hi-
stone hyperacetylation, and consequently, changes in gene
expression that can occur in a small proportion of genes
(10% or less) [35-38] or in more than 40% of genes [32].
However, selective HDAC8 inhibitors showed a unique pat-
tern of hyperacetylated proteins compared to the pan-HDAC
inhibitor Trichostatin A in human cells [39]. The “epigenetic
vulnerability” of certain cancer cells was proposed by Daw-
son and Kouzarides in 2012 as a cause of the relative speci-
ficity of HDACIs for cancer cells. The hypothesis proposed
that normal cells have alternative compensating pathways,
whereas cancer cells have a particular set of “essential”
genes that are regulated by epigenetic components, allowing
epigenetic inhibitors to disrupt the expression of a few key
target genes, leading to catastrophic effects in malignant
cells [40].
Epigenetic Evaluation of N-(2-hydroxyphenyl)-2-Propylpentanamide Current Molecular Pharmacology, 2020, Vol. 13, No. 0 5

Table 1. Oligonucleotides used to evaluate the expression of some genes associated with the arrest of the cell cycle and with epigenet-
ic mechanisms.

Gene Forward Reverse Size (bp)

1 p21 5’-AgACTCTCAgggTCgAAAAC-3’ 5’-ATTAgggCTTCCTCTTggAg-3’ 92
2 HDAC8 5’-gTCCCgAgTATgTCAgTATg -3’ 5’-CTATCCTCATCTgCTTATgC-3’ 108
3 GAPDH 5’-gTATgACAACAgCCTCAAgAT-3’ 5’-gTCCTTCCACgATACCAAAg-3’ 104

Fig. (1). VPA and VPA aryl derivative: N-(2-hydroxyphenyl)-2-propylpentamide (o-OH-VPA). (A higher resolution / colour version of this
figure is available in the electronic copy of the article).

Fig. (2). Effects of VPA and o-OH-VPA on the survival (A) and viability (B) of HeLa cells. Cells were incubated for 48 h at different concen-
trations of the compounds; the cell survival and viability were analyzed by MTT assay and the Trypan-blue exclusion method, respectively.
Data are expressed as the mean ± SD for n ≥ 6 wells from at least two independent experiments for cell survival and n = 4 for viability. (A
higher resolution / colour version of this figure is available in the electronic copy of the article).
6 Current Molecular Pharmacology, 2020, Vol. 13, No. 0 Luna-Palencia et al.

Fig. (3). Live cells per well, their doubling time (Dt) (A) and viability (B) of HeLa cells after being treated with 5 mM VPA and 0.1 mM o-O-
H-VPA for 24, 48 and 72 h as determined by the Trypan-blue exclusion method. Data are expressed as the mean ± SD for n = 3. (A higher re-
solution / colour version of this figure is available in the electronic copy of the article).

Fig. (4). Effects of 5 mM VPA and 0.1 mM o-OH-VPA on HDAC activity. Nuclear extracts from HeLa cells with HDAC activity were incu-
bated with 0.5% DMSO as a control and with the compounds. The percentage of HDAC activity is expressed as the mean ± SD for n = 3
from three independent experiments. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

Fig. (5). Expression of the HDAC8 gene by PCR (A) and qPCR (B) of HeLa cells treated with 5 mM VPA and 0.1 mM o-OH-VPA for 24 h.
The relative fold expression is expressed as the mean ± SD for n = 3 from three independent experiments. (A higher resolution / colour ver-
sion of this figure is available in the electronic copy of the article).
Epigenetic Evaluation of N-(2-hydroxyphenyl)-2-Propylpentanamide Current Molecular Pharmacology, 2020, Vol. 13, No. 0 7

Fig. (6). Expression of the p21 gene related to the arrest of the cell cycle (A), as well as the flow cytometry analysis of the cell cycle and
apoptosis (B) and the activity of caspase-3, where 1 µM Camptothecin was used as a positive control (C). Data are expressed as the mean ±
SD for n = 3 from three independent experiments. (A higher resolution / colour version of this figure is available in the electronic copy of the
article).

Fig. (7). Effect of 5 mM VPA and 0.1 mM o-OH-VPA on human fibroblast (FB) cell survival. Fibroblasts were incubated for 48 h with 0.5%
DMSO as a control and with the compounds at a concentration close to the IC50 obtained from assays with HeLa cells. The percentage of cell
survival was determined by the MTT assay and was expressed as the mean ± SD for n = 8 from two independent experiments. (A higher reso-
lution / colour version of this figure is available in the electronic copy of the article).

CONCLUSION effective than VPA. Furthermore, o-OH-VPA is more selec-

This contribution aimed to describe the action mech- tive than VPA and the results suggested in this study, as it
anism and selectivity of o-OH-VPA toward cancer cells. targets HDAC8. Similar to other HDACIs, o-OH-VPA has
The novel HDAC inhibitor o-OH-VPA exhibits HDAC in- anti-proliferative properties toward cancer cells but shows
hibitory effects at the µM scale and is fifty-eight times more no toxicity toward normal human fibroblasts. The inhibited
8 Current Molecular Pharmacology, 2020, Vol. 13, No. 0 Luna-Palencia et al.

proliferation of HeLa cell cultures by o-OH-VPA functions [8] de Ruijter, A.J.; van Gennip, A.H.; Caron, H.N.; Kemp, S.; van
via arresting the cell cycle in the G0/G1 phase, which occurs Kuilenburg, A.B.P. Histone deacetylases (HDACs): characteriza-
tion of the classical HDAC family. Biochem. J., 2003, 370(Pt 3),
as a result of the overexpression of p21 without caspase-3 ac- 737-749.[http://dx.doi.org/10.1042/bj20021321] [PMID:
tivation. 12429021]
[9] Gregoretti, I.V.; Lee, Y.M.; Goodson, H.V. Molecular evolution
ETHICS APPROVAL AND CONSENT TO PARTICI- of the histone deacetylase family: functional implications of phylo-
genetic analysis. J. Mol. Biol., 2004, 338(1),
PATE 17-31.[http://dx.doi.org/10.1016/j.jmb.2004.02.006] [PMID:
Not applicable. 15050820]
[10] Khan, N.; Jeffers, M.; Kumar, S.; Hackett, C.; Boldog, F.;
Khramtsov, N.; Qian, X.; Mills, E.; Berghs, S.C.; Carey, N.; Finn,
HUMAN AND ANIMAL RIGHTS P.W.; Collins, L.S.; Tumber, A.; Ritchie, J.W.; Jensen, P.B.;
No animals/humans were used for studies that are the ba- Lichenstein, H.S.; Sehested, M. Determination of the class and iso-
form selectivity of small-molecule histone deacetylase inhibitors.
sis of this research. Biochem. J., 2008, 409(2),
581-589.[http://dx.doi.org/10.1042/BJ20070779] [PMID:
CONSENT FOR PUBLICATION 17868033]
[11] Komulainen, T.; Lodge, T.; Hinttala, R.; Bolszak, M.; Pietilä, M.;
Not applicable. Koivunen, P.; Hakkola, J.; Poulton, J.; Morten, K.J.; Uusimaa, J.
Sodium valproate induces mitochondrial respiration dysfunction
AVAILABILITY OF DATA AND MATERIALS in HepG2 in vitro cell model. Toxicology, 2015, 331,
47-56.[http://dx.doi.org/10.1016/j.tox.2015.03.001] [PMID:
Not applicable. 25745980]
[12] Van den Wyngaert, I.; de Vries, W.; Kremer, A.; Neefs, J.; Verhas-
FUNDING selt, P.; Luyten, W.H.; Kass, S.U. Cloning and characterization of
human histone deacetylase 8. FEBS Lett., 2000, 478(1-2),
We gratefully acknowledge CONACYT (Grants: 77-83.[http://dx.doi.org/10.1016/S0014-5793(00)01813-5] [P-
CB-254600 and APN-782), Instituto Politécnico Nacional MID: 10922473]
[13] Oehme, I.; Deubzer, H.E.; Wegener, D.; Pickert, D.; Linke, J.P.;
(Grant: Proyectos Insignia IPN-2015), and CO- Hero, B.; Kopp-Schneider, A.; Westermann, F.; Ulrich, S.M.; von
FAA-SIP/IPN. Deimling, A.; Fischer, M.; Witt, O. Histone deacetylase 8 in neu-
roblastoma tumorigenesis. Clin. Cancer Res., 2009, 15(1),
CONFLICT OF INTEREST 91-99.[http://dx.doi.org/10.1158/1078-0432.CCR-08-0684] [P-
MID: 19118036]
The authors have no conflicts of interest, financial or [14] Luna-Palencia, G.R.; Martínez-Ramos, F.; Vásquez-Moctezuma,
otherwise. I.; Fragoso-Vazquez, M.J.; Mendieta-Wejebe, J.E.; Padilla--
Martínez, I.I.; Sixto-López, Y.; Méndez-Luna, D.; Trujillo-Fer-
rara, J.; Meraz-Ríos, M.A.; Fonseca-Sabater, Y.; Correa-Basurto,
ACKNOWLEDGEMENTS J. Three amino acid derivatives of valproic acid: design, synthesis,
The authors wish to thank M. Sc. Rosales-García V. H. theoretical and experimental evaluation as anticancer agents. Anti-
cancer. Agents Med. Chem., 2014, 14(7),
from CINVESTAV-IPN for his technical support in the cell 984-993.[http://dx.doi.org/10.2174/187152061466614012711321
cycle analysis and to Dr. Efraín Garrido, CINVESTAV-IPN 8] [PMID: 24476311]
for providing the HeLa cells. [15] Martínez-Ramos, F.; Luna-Palencia, G.R.; Vásquez-Moctezuma,
I.; Méndez-Luna, D.; Fragoso-Vázquez, M.J.; Trujillo-Ferrara, J.;
REFERENCES Meraz-Ríos, M.A.; Mendieta-Wejebe, J.E.; Correa-Basurto, J.
Docking Studies of Glutamine Valproic Acid Derivative (S)-5-
[1] Chen, H.P.; Zhao, Y.T.; Zhao, T.C. Histone deacetylases and amino-2-(heptan-4-ylamino)-5-oxopentanoic Acid (Gln-VPA) on
mechanisms of regulation of gene expression. Crit. Rev. Oncog., HDAC8 with Biological Evaluation in HeLa Cells. Anticancer.
2015, 20(1-2), Agents Med. Chem., 2016, 16(11),
35-47.[http://dx.doi.org/10.1615/CritRevOncog.2015012997] [P- 1485-1490.[http://dx.doi.org/10.2174/1871520616666160204111
MID: 25746103] 158] [PMID: 26845132]
[2] Wolffe, A.P. Chromatin: Structure and Function., (3rd ed. ), 3rd [16] Luna-Palencia, G.R. Efecto de los inhibidores de las desacetilasas
ed. 1998, de histonas sobre la expresión de genes de resistencia a drogas en
[3] Li, T.; Song, B.; Wu, Z.; Lu, M.; Zhu, W.G. Systematic identifica- la línea celular HeLa., 2015.
tion of Class I HDAC substrates. Brief. Bioinform., 2014, 15(6), [17] Prestegui-Martel, B.; Bermúdez-Lugo, J.A.; Chávez-Blanco, A.;
963-972.[http://dx.doi.org/10.1093/bib/bbt060] [PMID: Dueñas-González, A.; García-Sánchez, J.R.; Pérez-González,
23975722] O.A.; Padilla-Martínez, I.I.; Fragoso-Vázquez, M.J.; Mendie-
[4] Hellebrekers, D.M.; Griffioen, A.W.; van Engeland, M. Dual tar- ta-Wejebe, J.E.; Correa-Basurto, A.M.; Méndez-Luna, D.; Trujil-
geting of epigenetic therapy in cancer. Biochim. Biophys. Acta, lo-Ferrara, J.; Correa-Basurto, J. N-(2-hydrox-
2007, 1775(1), 76-91.[PMID: 16930846] yphenyl)-2-propylpentanamide, a valproic acid aryl derivative de-
[5] Yoo, C.B.; Jones, P.A. Epigenetic therapy of cancer: past, present signed in silico with improved anti-proliferative activity in HeLa,
and future. Nat. Rev. Drug Discov., 2006, 5(1), rhabdomyosarcoma and breast cancer cells. J. Enzyme Inhib. Med.
37-50.[http://dx.doi.org/10.1038/nrd1930] [PMID: 16485345] Chem., 2016, 31(sup3),
[6] Minucci, S.; Pelicci, P.G. Histone deacetylase inhibitors and the 140-149.[http://dx.doi.org/10.1080/14756366.2016.1210138] [P-
promise of epigenetic (and more) treatments for cancer. Nat. Rev. MID: 27483122]
Cancer, 2006, 6(1), 38-51.[http://dx.doi.org/10.1038/nrc1779] [P- [18] Roth, V. 2006.Doubling Time Computing,
MID: 16397526] http://www.doubling-time.com/compute.php
[7] Witt, O.; Deubzer, H.E.; Milde, T.; Oehme, I. HDAC family: [19] Pfaffl, M.W. A new mathematical model for relative quantifica-
What are the cancer relevant targets? Cancer Lett., 2009, 277(1), tion in real-time RT-PCR. Nucleic Acids Res., 2001,
8-21.[http://dx.doi.org/10.1016/j.canlet.2008.08.016] [PMID: 29(9)e45[http://dx.doi.org/10.1093/nar/29.9.e45] [PMID:
18824292] 11328886]
Epigenetic Evaluation of N-(2-hydroxyphenyl)-2-Propylpentanamide Current Molecular Pharmacology, 2020, Vol. 13, No. 0 9

[20] Contis-Montes de Oca, A.; Rodarte-Valle, E.; Rosales-Hernández, 1191-1200.[http://dx.doi.org/10.7164/antibiotics.53.1191] [PMID:

M.C.; Abarca-Rojano, E.; Rojas-Hernández, S.; Fragoso-Vázquez, 11132966]
M.J.; Mendieta-Wejebe, J.E.; Correa-Basurto, A.M.; Vázquez-- [30] Peart, M.J.; Tainton, K.M.; Ruefli, A.A.; Dear, A.E.; Sedelies,
Moctezuma, I.; Correa-Basurto, J. N-(2′-Hydrox- K.A.; O’Reilly, L.A.; Waterhouse, N.J.; Trapani, J.A.; Johnstone,
yphenyl)-2-propylpentanamide (OH-VPA), a histone deacetylase R.W. Novel mechanisms of apoptosis induced by histone deacety-
inhibitor, induces the release of nuclear HMGB1 and modifies lase inhibitors. Cancer Res., 2003, 63(15), 4460-4471.[PMID:
ROS levels in HeLa cells. Oncotarget, 2018, 9(70), 12907619]
33368-33381.[http://dx.doi.org/10.18632/oncotarget.26077] [P- [31] Childs, B.G.; Baker, D.J.; Kirkland, J.L.; Campisi, J.; van
MID: 30279967] Deursen, J.M. Senescence and apoptosis: dueling or complemen-
[21] Hu, E.; Chen, Z.; Fredrickson, T.; Zhu, Y.; Kirkpatrick, R.; tary cell fates? EMBO Rep., 2014, 15(11),
Zhang, G.F.; Johanson, K.; Sung, C.M.; Liu, R.; Winkler, J. Clon- 1139-1153.[http://dx.doi.org/10.15252/embr.201439245] [PMID:
ing and characterization of a novel human class I histone deacety- 25312810]
lase that functions as a transcription repressor. J. Biol. Chem., [32] Peart, M.J.; Smyth, G.K.; van Laar, R.K.; Bowtell, D.D.; Richon,
2000, 275(20), V.M.; Marks, P.A.; Holloway, A.J.; Johnstone, R.W. Identifica-
15254-15264.[http://dx.doi.org/10.1074/jbc.M908988199] [P- tion and functional significance of genes regulated by structurally
MID: 10748112] different histone deacetylase inhibitors. Proc. Natl. Acad. Sci.
[22] Waltregny, D.; De Leval, L.; Glénisson, W.; Ly Tran, S.; North, USA, 2005, 102(10),
B.J.; Bellahcène, A.; Weidle, U.; Verdin, E.; Castronovo, V. Ex- 3697-3702.[http://dx.doi.org/10.1073/pnas.0500369102] [PMID:
pression of histone deacetylase 8, a class I histone deacetylase, is 15738394]
restricted to cells showing smooth muscle differentiation in nor- [33] Vanaja, G.R.; Ramulu, H.G.; Kalle, A.M. Overexpressed HDAC8
mal human tissues. Am. J. Pathol., 2004, 165(2), in cervical cancer cells shows functional redundancy of tubulin
553-564.[http://dx.doi.org/10.1016/S0002-9440(10)63320-2] [P- deacetylation with HDAC6. Cell Commun. Signal., 2018, 16(1),
MID: 15277229] 20.[http://dx.doi.org/10.1186/s12964-018-0231-4] [PMID:
[23] Balasubramanian, S.; Ramos, J.; Luo, W.; Sirisawad, M.; Verner, 29716651]
E.; Buggy, J.J. A novel histone deacetylase 8 (HDAC8)-specific [34] Korfei, M.; Stelmaszek, D.; MacKenzie, B.; Skwarna, S.; Chillap-
inhibitor PCI-34051 induces apoptosis in T-cell lymphomas. pagari, S.; Bach, A.C.; Ruppert, C.; Saito, S.; Mahavadi, P.;
Leukemia, 2008, 22(5), Klepetko, W.; Fink, L.; Seeger, W.; Lasky, J.A.; Pullamsetti, S.S.;
1026-1034.[http://dx.doi.org/10.1038/leu.2008.9] [PMID: Krämer, O.H.; Guenther, A. Comparison of the antifibrotic effects
of the pan-histone deacetylase-inhibitor panobinostat versus the
18256683]
IPF-drug pirfenidone in fibroblasts from patients with idiopathic
[24] Vannini, A.; Volpari, C.; Filocamo, G.; Casavola, E.C.; Brunetti,
pulmonary fibrosis. PLoS One, 2018,
M.; Renzoni, D.; Chakravarty, P.; Paolini, C.; De Francesco, R.;
13(11)e0207915[http://dx.doi.org/10.1371/journal.pone.0207915]
Gallinari, P.; Steinkühler, C.; Di Marco, S. Crystal structure of a
[PMID: 30481203]
eukaryotic zinc-dependent histone deacetylase, human HDAC8,
[35] Van Lint, C.; Emiliani, S.; Verdin, E. The expression of a small
complexed with a hydroxamic acid inhibitor. Proc. Natl. Acad. fraction of cellular genes is changed in response to histone hyper-
Sci. USA, 2004, 101(42), acetylation. Gene Expr., 1996, 5(4-5), 245-253.[PMID: 8723390]
15064-15069.[http://dx.doi.org/10.1073/pnas.0404603101] [P- [36] Glaser, K.B.; Staver, M.J.; Waring, J.F.; Stender, J.; Ulrich, R.G.;
MID: 15477595] Davidsen, S.K. Gene expression profiling of multiple histone
[25] Yang, Z.Y.; Perkins, N.D.; Ohno, T.; Nabel, E.G.; Nabel, G.J. The deacetylase (HDAC) inhibitors: defining a common gene set pro-
p21 cyclin-dependent kinase inhibitor suppresses tumorigenicity duced by HDAC inhibition in T24 and MDA carcinoma cell lines.
in vivo. Nat. Med., 1995, 1(10), Mol. Cancer Ther., 2003, 2(2), 151-163.[PMID: 12589032]
1052-1056.[http://dx.doi.org/10.1038/nm1095-1052] [PMID: [37] Mitsiades, C.S.; Mitsiades, N.S.; McMullan, C.J.; Poulaki, V.;
7489362] Shringarpure, R.; Hideshima, T.; Akiyama, M.; Chauhan, D.; Mun-
[26] Halsall, J.A.; Turan, N.; Wiersma, M.; Turner, B.M. Cells adapt to shi, N.; Gu, X.; Bailey, C.; Joseph, M.; Libermann, T.A.; Richon,
the epigenomic disruption caused by histone deacetylase inhibi- V.M.; Marks, P.A.; Anderson, K.C. Transcriptional signature of hi-
tors through a coordinated, chromatin-mediated transcriptional re- stone deacetylase inhibition in multiple myeloma: biological and
sponse. Epigenetics Chromatin, 2015, 8, clinical implications. Proc. Natl. Acad. Sci. USA, 2004, 101(2),
29.[http://dx.doi.org/10.1186/s13072-015-0021-9] [PMID: 540-545.[http://dx.doi.org/10.1073/pnas.2536759100] [PMID:
26380582] 14695887]
[27] Archer, S.Y.; Meng, S.; Shei, A.; Hodin, R.A. p21(WAF1) is re- [38] Dejligbjerg, M.; Grauslund, M.; Litman, T.; Collins, L.; Qian, X.;
quired for butyrate-mediated growth inhibition of human colon Jeffers, M.; Lichenstein, H.; Jensen, P.B.; Sehested, M. Differen-
cancer cells. Proc. Natl. Acad. Sci. USA, 1998, 95(12), tial effects of class I isoform histone deacetylase depletion and en-
6791-6796.[http://dx.doi.org/10.1073/pnas.95.12.6791] [PMID: zymatic inhibition by belinostat or valproic acid in HeLa cells.
9618491] Mol. Cancer, 2008, 7,
[28] Richon, V.M.; Sandhoff, T.W.; Rifkind, R.A.; Marks, P.A. His- 70.[http://dx.doi.org/10.1186/1476-4598-7-70] [PMID: 18789133]
tone deacetylase inhibitor selectively induces p21WAF1 expres- [39] K.; Marshall, B.L.; Hedglin, M.; Verdin, E.; Ulrich, S.M. Design
sion and gene-associated histone acetylation. Proc. Natl. Acad. and evaluation of ʻLinkerlessʼ hydroxamic acids as selective
Sci. USA, 2000, 97(18), HDAC8 inhibitors. Bioorg. Med. Chem. Lett., 2007, 17,
10014-10019.[http://dx.doi.org/10.1073/pnas.180316197] [PMID: 2874-2878.[http://dx.doi.org/10.1016/j.bmcl.2007.02.064]
10954755] [40] Dawson, M.A.; Kouzarides, T. Cancer epigenetics: from mech-
[29] Kim, Y.B.; Ki, S.W.; Yoshida, M.; Horinouchi, S. Mechanism of anism to therapy. Cell, 2012, 150(1),
cell cycle arrest caused by histone deacetylase inhibitors in human 12-27.[http://dx.doi.org/10.1016/j.cell.2012.06.013] [PMID:
carcinoma cells. J. Antibiot. (Tokyo), 2000, 53(10), 22770212]