Discovery of N-(2-Amino-4-Fluorophenyl)-4-[bis-(2-Chloroethyl)-Amino]-Benzamide as a Potent HDAC3 Inhibitor

Article abstract of DOI:10.3389/fonc.2020.592385

In discovery of HDAC inhibitors with improved activity and selectivity, fluorine substitution was performed on our previously derived lead compound. The synthesized molecules N-(2-amino-4-fluorophenyl)-4-[bis-(2-chloroethyl)-amino]-benzamide (FNA) exhibited class I (HDAC1, 2, and 3) selectivity in the in vitro enzymatic assay and especially potent against HDAC3 activity (IC₅₀: 95.48 nM). The results of in vitro antiproliferative assay indicated that FNA exhibited solid tumor cell inhibitory activities with IC₅₀ value of 1.30 μM against HepG2 cells compared with SAHA (17.25 μM). Moreover, the in vivo xenograft model study revealed that FNA could inhibit tumor growth with tumor growth inhibition (TGI) of 48.89% compared with SAHA (TGI of 48.13%). Further HepG2 cell–based apoptosis and cell cycle studies showed that promotion of apoptosis and G2/M phase arrest make contributions to the antitumor activity of FNA. In addition, drug combination results showed that 0.5 μM of FNA could improve the anticancer activity of taxol and camptothecin. The present studies revealed the potential of FNA utilized as a high potent lead compound for further discovery of isoform selective HDAC inhibitors.

Full text of DOI:10.3389/fonc.2020.592385

ORIGINAL RESEARCH
published: 15 October 2020
doi: 10.3389/fonc.2020.592385
Discovery of
N-(2-Amino-4-Fluorophenyl)-4-[bis-
(2-Chloroethyl)-Amino]-Benzamide
as a Potent HDAC3 Inhibitor
1
1
1
1
Yiming Chen1†, Jinhong Feng2†, Yajie Hu , Xuejian Wang , Weiguo Song and Lei Zhang
*
*
1 Department of Medicinal Chemistry, School of Pharmacy, Weifang Medical University, Weifang, China, 2 Key Laboratory for
Applied Technology of Sophisticated Analytical Instruments of Shandong Province, Analysis and Test Center, Qilu University
of Technology (Shandong Academy of Sciences), Jinan, China
In discovery of HDAC inhibitors with improved activity and selectivity, fluorine
substitution was performed on our previously derived lead compound. The synthesized
molecules N-(2-amino-4-fluorophenyl)-4-[bis-(2-chloroethyl)-amino]-benzamide (FNA)
exhibited class I (HDAC1, 2, and 3) selectivity in the in vitro enzymatic assay and
especially potent against HDAC3 activity (IC50: 95.48 nM). The results of in vitro
antiproliferative assay indicated that FNA exhibited solid tumor cell inhibitory activities with
IC50 value of 1.30 µM against HepG2 cells compared with SAHA (17.25 µM). Moreover,
the in vivo xenograft model study revealed that FNA could inhibit tumor growth with tumor
growth inhibition (TGI) of 48.89% compared with SAHA (TGI of 48.13%). Further HepG2
cell–based apoptosis and cell cycle studies showed that promotion of apoptosis and
G2/M phase arrest make contributions to the antitumor activity of FNA. In addition, drug
combination results showed that 0.5 µM of FNA could improve the anticancer activity of
taxol and camptothecin. The present studies revealed the potential of FNA utilized as a
high potent lead compound for further discovery of isoform selective HDAC inhibitors.
Edited by:
Zhe-Sheng Chen,
St. John’s University, United States
Reviewed by:
Li Tan,
University of Michigan, United States
Junjie Zhu,
University of Pittsburgh, United States
*Correspondence:
Weiguo Song
songwg@139.com
Lei Zhang
leizhangchemical@gmail.com
†These authors have contributed
equally to this work
Keywords: 4-fluorine-benzamide, nitrogen mustard, HDAC, antitumor activity, isoform selectivity
INTRODUCTION
Specialty section:
This article was submitted to
Pharmacology of Anti-Cancer Drugs,
a section of the journal
Histone deacetylases and histone acetylases are important enzymes participating in the regulation
of gene expression by acetylating and deacetylating of histones (1, 2). Specifically, HDACs are the
enzymes controlling the epigenetic modifications of histone, along with more than 50 nonhistone
proteins (3, 4). So far, a total of 18 different HDACs isoforms have been identified and classified
into four classes according to their size, distribution in cells, and homology (5–8). Among the four
classes, classes I (HDAC1, 2, 3, and 8), II (HDAC4, 5, 6, 7, 9, and 10), and IV (HDAC11) HDACs
require zinc ion as cofactor and thus are known as zinc-dependent enzymes. On the other hand,
class III HDACs are a group of NAD+-dependent enzymes (also known as sirtuins), whose activity
does not require the presence of zinc iron (9–13).
In recent years, inhibition of HDACs has exhibited potency for the treatment tumors
(14, 15), diabetes (16), Parkinson disease (17), inflammation (18, 19), HIV (20), and heart
disease (21). In tumor cells, it had been shown that overexpression of HDACs led to increased
deacetylation of histones, which increases the gravitational pull between DNA and histones by
Frontiers in Oncology
Received: 06 August 2020
Accepted: 01 September 2020
Published: 15 October 2020
Citation:
Chen Y, Feng J, Hu Y, Wang X,
Song W and Zhang L (2020) Discovery
of N-(2-Amino-4-Fluorophenyl)-4-[bis-
(2-Chloroethyl)-Amino]-Benzamide as
a Potent HDAC3 Inhibitor.
Front. Oncol. 10:592385.
doi: 10.3389/fonc.2020.592385
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Nitrogen Mustard HDAC Inhibitor
restoring the positive charge of the histones, making the relaxed
nucleosomes very tight and unfavorable for the expression of
specific genes, including some tumor suppressor genes (22–28).
In the field of epigenetics, HDAC inhibitors (HDACIs) have
been successfully developed in the antitumor therapy, and
several HDACIs have been developed into the market (29).
Vorinostat (SAHA) is the first approved HDACI, which has
been administered clinically for the treatment of cutaneous T-
cell lymphoma (CTCL) (30). Afterward, romidepsin (FK-228),
belinostat (PXD101), and panobinostat (LBH589) were approved
for the treatment of CTCL, peripheral T-cell lymphoma (PTCL),
and multiple myeloma, respectively (31–33). Chidamide (CS055)
was approved by the Chinese Food and Drug Administration
for the treatment of PTCL (34). Generally, pharmacophores
of HDACIs are consist of three structural elements: a capping
group, which recognizes the hydrophobic region at the opening
of HDAC active site; a linker, which connects the hydrophobic
ring and the zinc-binding group (ZBG) via occupation of the
tubular channel; a ZBG, whose functions include binding to
the zinc ion located in the active center of HDACs, as well as
forming hydrogen bonds with certain amino acid residues of
active sites (35–37).
clinical because of its relatively low toxicity such as chlormethine
(40), chlorambucil (41), and melphalan (42).
In discovery of novel and potent HDACIs, aromatic nitrogen
mustard parts were integrated into the structure of HDACI
CI994 in our previous study (43). The resulting molecule, N-
(2-aminophenyl)-4-(bis(2-chloroethyl)amino)benzamide (NA)
exhibited class I selectivity in the enzymatic assay and potent
in vitro antitumor activity in the cell based assay. However, NA
exhibited lower potency than SAHA in the in vivo assay using
nude mice xenograft model with inoculation of HepG2 cells.
Fluorine substitution in the benzamide ZBG was discovered to
improve the metabolic stability of HDACIs, such as the design
of chidamide (44, 45). In the present study, to improve the
selectivity, activity, and in vivo stability of NA, fluorine was
introduced to the para-position of amide bond in the phenyl
ring of ZBG considering the structure of chidamide (Figure 1).
The designed compound N-(2-amino-4-fluorophenyl)-4-[bis-
(2-chloroethyl)-amino]-benzamide (FNA) was synthesized and
evaluated in the antitumor assay.
RESULTS AND DISCUSSION
Nitrogen mustard anticancer drugs were used clinically since Chemistry
1942, which effectively bind and cross-link to DNA, resulting
in prevention of DNA replication and cell proliferation (38).
Nitrogen mustard antitumor drugs are mainly composed of
alkylation part and carrier part. According to different carriers,
they can be divided into aliphatic nitrogen mustard and aromatic
nitrogen mustard (39). Aromatic nitrogen mustard is still used in
The designed compound FNA was synthesized as described
in Scheme 1. Methyl esterification was performed to protect
the starting material 4-aminobenzoic acid (a). To synthesize
intermediate methyl 4-(bis(2-hydroxyethyl)amino)benzoate (c),
2-hydroxyethyl groups were added by reaction of intermediate
b with ethylene oxide. Subsequent chlorine substitution and
FIGURE 1 | The design of FNA.
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SCHEME 1 | (1) CH3COCl, MeOH, reflux 5 h. (2) oxirane, H2O/CH3COOH, 0◦C 48 h. (3) POCl3, MB, reflux 4 h. (4) 4 M HCl, reflux 4 h. (5) 1,2-diamino-4-
fluorobenzene, CDI, THF (dry), rt overnight.
TABLE 1 | Enzyme inhibitory activity of FNA compared with MS275 and SAHA
(IC50, nM)a.
and HDAC2, respectively. The results suggested that FNA has the
potential to be utilized as a lead compound for the discovery of
HDAC3-selective inhibitors.
Among all the HDAC isoforms found in human, HDAC3
HDACs HDAC1 HDAC2 HDAC3 HDAC4 HDAC6 HDAC7 HDAC8 HDAC9
is unique for its expression in the nucleus, cytoplasm, or
membrane. As a single HDAC isoform, HDAC3 was revealed
to promote the phosphorylation and activation of AKT, which
specifically binds to HDAC3, participate in the self-renewal of
liver cancer stem cells, and engage in the growth of triple-negative
breast cancer cells (15). Therefore, discovery of selective HDAC3
inhibitors make contributions to the treatment of specific
diseases related to the abnormal function of HDAC3. As a potent
lead compound, FNA could be utilized for further structural
modification in discovery of HDAC3-selective inhibitors.
FNA
MS275 46.17 100.90 43.89 >5,000 >5,000 >5,000 >5,000 >5,000
SAHA 52.90 90.78 167.24 >5,000 172.10 >5,000 4,120 >5,000
842.80 949.15 95.48 >5,000 >5,000 >5,000 >5,000 >5,000
aAssays were performed in replicate (n ≥ 2), the SD values are <10% of the mean.
deprotection of carboxyl group afforded key intermediate 4-
(bis(2-chloroethyl)amino)benzoic acid (e). Target compound
FNA was synthesized by condensation of intermediate e with 1,2-
diamino-4-fluorobenzene.
Enzyme Inhibitory Selectivity of FNA
Antiproliferative Activity of FNA
To assess the isoform selectivity and the inhibitory activity of the
derived FNA, enzymatic assay was performed against HDAC1,
2, 3, 4, 6, 7, 8, and 9 using SAHA (nonselective inhibitor) and
MS275 (class I selective inhibitor) as positive control drugs. The
selectivity of isoforms and IC50 of the tested compounds were
displayed in Table 1. According to the results, FNA exhibited
IC50 values of 842.80, 949.15, and 95.48 nM against HDAC1, 2,
and 3, respectively. While in inhibition of HDAC4, 6, 7, 8, and 9,
FNA exhibited more than 5,000 nM of IC50 values. It is suggested
that FNA is a highly class I–selective inhibitor. Nevertheless, in
the inhibition of HDAC1, 2, and 3, it is remarkable that FNA
showed 8.83- and 9.94-fold of HDAC3 selectivity vs. HDAC1
The in vitro antiproliferative activities of target compound
FNA along with the positive control SAHA were tested against
multiple tumor cell lines, including the lung cancer (H460, H322,
and A549), colon carcinoma SW480, renal carcinoma (OS-RC-
2, SK-NEP-1), thyroid cancer (FTC-133, SW-579), breast cancer
(MDA-MB-231), ovarian cancer (A2780), cervical carcinoma
(Hela), myeloma (U266), liver cancer (HepG2), and leukemic
(U937 and K562) cells. According to the results shown in Table 2,
potent antiproliferative activities against most of the tumor cell
lines tested (except SW480 and OR-RC-2) were observed from
FNA, as evidenced by the low IC50 values. Compared with SAHA,
it is obvious that FNA could effectively inhibit the growth of
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HepG2, U937, H460, FTC-133, HELA, and K562 cells with IC50
values of 1.30, 0.55, 4.73, 9.09, 1.41, and 1.31 µM, respectively. It
showed that FNA has a significant inhibitory effect on both solid
tumor cells and nonsolid tumor cells. Remarkably, in inhibition
the growth of HepG2 cells, FNA (similar to NA) was revealed
to be 13.3-fold (IC50 value of 1.30 µM) more potent relative to
SAHA, whose IC50 value is 17.25 µM. The present results indicate
the future of development of FNA analogs for the treatment of
liver cancer.
TABLE 2 | Antiproliferative activities of compound FNA against human cancer
cells (IC50, µM)a.
In vivo Antitumor Activity
Cell line
FNA (µM)
SAHA (µM)
To further investigate the anticancer activity of FNA, HepG2
xenograft nude mice model was utilized to assess the in vivo
antitumor activity of compound FNA. Mice were injected
intraperitoneally with FNA and SAHA, both at 100 mg/kg, once
a day for 15 days. When the tumor is prominent, the BALB/c
female mice were randomly assigned into control and treatment
groups (six mice per group). As shown in Figure 2A, all the mice
in the treatment groups displayed no significant change in body
weight. The results showed that both FNA and SAHA can inhibit
tumor growth compared with the control group (Figures 2B–D).
Compound FNA effectively inhibited the tumor growth with
tumor growth inhibition (TGI) rate of 48.89% compared with
SAHA (with TGI of 48.13%). Although FNA exhibited improved
inhibitory activity compared with SAHA in the in vitro test,
the activity improvement was not obvious in the in vivo study.
It is suggested that further structural modification of FNA is
needed to improve the activity and pharmacokinetic properties.
To improve the in vivo activity of FNA, introduction of bioactive
HepG2
U937
1.30 0.25
0.55 0.03
4.73 0.05
>100
17.25 0.46
0.86 0.03
7.63 0.03
2.91 0.04
>100
H460
SW480
OS-RC-2
H322
>100
6.36 0.07
8.32 0.18
9.09 0.13
52.40 0.13
35.29 0.03
33.74 0.04
4.30 0.12
1.41 0.04
1.31 0.05
0.63 0.32
2.54 0.06
3.68 0.02
24.30 0.10
24.30 0.07
5.82 0.08
4.92 0.04
2.71 0.09
1.89 0.05
2.52 0.05
0.22 0.03
SK-NEP-1
FTC-133
SW579
MDA-MB-231
A549
A2780
Hela
K562
U266
aAssays were performed in replicate (n ≥ 2).
FIGURE 2 | Results of in vivo evaluation of FNA in the HepG2 xenografts mouse model. (A) Body weight of mice. (B) Tumor volume measurements from HepG2
xenografts mice in each group. (C) The final tumor weights in each group. *p < 0.05 compared with the control group. (D) Images of the excised tumors in each group.
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FIGURE 3 | Proapoptotic effect of molecule FNA. HepG2 cells were exposed to FNA and SAHA at concentrations of 1, 3, and 9 µM for 24 h. Then cells were stained
with annexin V–FITC/PI, and the apoptotic status of the cells was assessed with flow cytometry analysis.
groups with anticancer activities to the structure of FNA will be
performed in further studies.
concentration increase from 0.125 to 0.5 µM) group. However,
at the tested concentrations, SAHA did not exhibit any effects
in the regulation of HepG2 cell cycle. The results indicated that
induction of the G2/M phase arrest also plays a significant role in
the antiproliferative effects of molecule FNA.
Cell Apoptosis Analysis
In order to confirm whether apoptosis contributes to the
observed antiproliferative activities of FNA, apoptosis study
was performed using HepG2 cells. Flow cytometry analysis
was shown in Figure 3. From the annexin V–fluorescein
isothiocyanate/propidium iodide (FITC/PI) stating data, it
is obvious that compound FNA promoted cell apoptosis
against HepG2 cells dose-dependently. Following treatment with
different doses of FNA (1, 3, and 9 µM), the apoptosis rate
of HepG2 cells was significantly elevated from 5.83% of the
normal group to 14.08, 19.11, and 28.83% compared with SAHA
(apoptosis rate of 10.03, 10.91, and 12.43% at concentrations of
1, 3, and 9 µM), respectively. It is suggested that apoptosis plays
a role in the HepG2 cell inhibitory activity of FNA.
Antiproliferative Activities of FNA in
Combination With Taxol and Camptothecin
It had been reported that HDACIs (HDACIs) may work as
chemosensitizers when used together with other antitumor drugs
(8). Because of the high cell cycle arrest ability of FNA, drug
combination investigation was performed by combining FNA
with the G2/M phase arrest drug taxol and camptothecin. HepG2
cells were used for the test, and percentage inhibition rate (PIR)
was used as a measure of potency. As shown in Figure 5, it is
revealed that the PIRs of FNA (0.5 µM) in combination with
taxol and camptothecin are higher than that of the single-drug
groups on HepG2 cells. The PIRs of taxol were 57.07 and 62.41
at the concentrations of 0.1 and 0.2 µM, respectively. Addition of
0.5 µM of FNA increased the PIR to 62.43 (0.1 µM of taxol) and
67.23 (0.2 µM of taxol), respectively. The PIRs of camptothecin
at doses of 0.25 and 0.5 µM were 61.70 and 60.86, respectively.
Improved activities were obtained by addition of FNA (0.5 µM)
with PIR values of 67.07 (0.25 µM of camptothecin) and 75.52
(0.5 µM of camptothecin), respectively. It is suggested that FNA
could synergistically improve the antiproliferative ability of the
cell cycle arrest drugs such as taxol and camptothecin.
Cell Cycle Analysis
Generally, the cell cycle consists of three phases: G0/G1, S,
and G2/M phase. A characteristic change in tumor cells is
dysregulated cell cycle due to genetic mutations, resulting in
uncontrolled cell proliferation. The designed compound FNA
was evaluated for the cell cycle effect on HepG2 with various
doses (0.125, 0.25, and 0.5 µM). As shown in Figure 4, it is
significant that FNA increased cell number at G2/M phase
with raising concentrations. The percentage of cells in G2/M
phase was increased from 18.84 to 59.36% in the FNA (with
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FIGURE 4 | Cell cycle analysis on HepG2 cells treated with FNA. Cells were treated with FNA and SAHA at concentrations of 0.125, 0.25, and 0.5 µM for 6 h. The
results were evaluated with flow cytometry analysis.
CONCLUSION
The dry THF was used by heating reflux with sodium. TLC
with 0.25-mm silica gel plates (60GF-254) was used to monitor
all the reactions. The sports were visualized with UV light
and ferric chloride. With a Burker DRX spectrometer, the
1H NMR spectra were recorded at 500 MHz, using TMS
as an internal standard. High-resolution mass spectra were
performed at Weifang Medical University in Weifang, China.
The derived target compound (FNA) is of 95.48% purity analyzed
by ultraperformance liquid chromatography (UPLC), which was
performed on a Waters Acquity H class UPLC instrument using
Benzamide HDACIs exhibited the advantage of class I selectivity
compared with HDACIs with hydroxamic acid as their ZBGs.
However, none of the benzamide HDACIs have been approved
by US Food and Drug Administration (FDA) yet. The
fluorine substituted benzamide HDACI, chidamide, approved
by the CFDA, exhibited advantage of high pharmacokinetic
properties compared with the unsubstituted benzamide (such
as MS275). Therefore, fluorine substituted was performed on
the previous lead compound NA. The derived FNA exhibited
HDAC3 selectivity and high HepG2 cell inhibitory activity.
Moreover, FNA was also effective in the HepG2 nude mice
xenograft model–based assay. Further investigations revealed
that promotion of apoptosis and cell cycle arrest at G2/M
phase both contributed to the antitumor activity of FNA.
Moreover, in combination with FNA, taxol and camptothecin
exhibited improved antiproliferative activities against HepG2
cells. Collectively, a potent HDAC3 inhibitor was discovered,
which could be utilized as a lead compound in the development
of new drugs for cancer treatments.
R
an Acquity UPLCꢀBEH C18 (150 × 2.1 mm). The mobile phase
was acetonitrile–water (90:10), and detection wavelength was
254 nm.
The
synthesis
and
description
of
4-(bis(2-
chloroethyl)amino)benzoic acid (e) were presented in our
previous work (43).
(2-Aminophenyl)-4-(bis(2-
Chloroethyl)Amino)Benzamide
To a solution of compound e (2.00 g, 7.7 mmol) in THF (50 mL),
CDI (1.87 g, 11.6 mmol) was added, and the solution was refluxed
for 3 h. 1,2-Diamino-4-fluorobenzene (3.8 g, 30.6 mmol) and
TFA (1.1 g, 9.24 mmol) were added with stirring, and the mixture
was kept for 16 h at room temperature. The solvent was then
evaporated with the residue being dissolved in EtOAc (50 mL).
The resulting EtOAc solution was washed with NaHCO3 (3 ×
20 mL), 1 M citric acid (3 × 20 mL), and brine (3 × 20 mL),
MATERIALS AND METHODS
Chemistry
All the starting materials and reagents commercially available
were used in the current study without further purifications.
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FIGURE 5 | Antiproliferative activities of taxol and camptothecin alone and in combination with FNA.
dried over MgSO4, and evaporated under vacuum. The desired
compound FNA was obtained by crystallization in EtOAc under
4◦C as brown powder. HRMS (AP-ESI) m/z calculated for
reaction was initiated by adding HDAC substrate working
solution and incubating at 30◦C for 2 h. After the desired time,
10 µL developer with trypsin and trichostatin A was added to
stop the reaction, and then the mixture was incubated at 30◦C
for another 30 min.
A microplate reader was used to determine fluorescence
intensity at excitation: 360 nm and emission: 460 nm. The
inhibition ratios were calculated by comparing the fluorescence
intensities from tested wells to those of controls. The IC50 curves
and values were then obtained with GraphPad Prism 6.0 software.
1
C17H19Cl2FN3O [M+H]+ 370.0889 found 370.0870. H NMR
1
(400 MHz, (CD3)2SO): δ = 9.33 (s, H), 7.86 (d, J = 8.8 Hz,
2H), 7.08 (dd, J1 = 2.3 Hz, J2 = 6.4 Hz, 1H), 6.83 (d, J = 8.8 Hz,
2H), 6.54 (dd, J1 = 2.6 Hz, J2 = 11.2 Hz, 1H), 6.35 (td, J1
=
2.8 Hz, J2 = 8.5 Hz, 1H), and 5.14 (s, 2H), 3.86–3.75 (m, 8H). 13
C
NMR (400 MHz, (CD3)2SO): δ = 165.60, 161.32, 149.41, 145.81,
130.06, 127.80, 122.59, 120.42, 111.39, 102.60, 102.09, 52.33, and
41.52 ppm.
Antiproliferative Activity of FNA
Enzyme Inhibitory Selectivity of FNA
Antiproliferative activities of FNA were evaluated with cell
viability assay (MTT assay) with SAHA as the control drug (46).
The stock solutions of compounds to be tested were prepared
in culture medium. Tumor cell lines were cultured in 96-well
plates at a density of 5 × 103 cells per well and incubated
until 90–95% confluence, and then each well received 100 µL
medium containing desired concentrations of test compounds,
and then incubated at 37◦C and 5% CO2 for 48 h. To determine
cell viability, 20 µL MTT working solution (5 mg/mL) was then
added to each well and incubated for another 4 h. After this
incubation, the medium was carefully aspirated, and 200 µL
All of the HDAC enzymes tested were purchased from BPS
Bioscience. First, 20 µL of each recombinant HDAC enzyme
solution (HDAC1, 2, 3, 4, 6, 7, 8, and 9) was mixed with various
concentrations of tested compound samples (20 µL) in a 96-
well plate. The mixture was incubated at 30◦C for 1 h for the
dose-dependent assay. Additionally, mixtures were incubated
for 15, 30, 60, and 90 min, for the time-dependent assay, and
then 10 µL of fluorogenic substrate [3 mM Boc-Lys(acetyl)-
AMC or Boc-Lys (trifluoroacetyl)-AMC for HDAC1/2/3/6 or
HDAC4/7/8/9, respectively] was added. Then, the acetylation
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dimethyl sulfoxide (DMSO) was added to each well and vibrated Drug Combination Analysis
for 10 min to make sure formed formazans were completely
dissolved. The optical densities (ODs) at 490 and 630 nm
were counted by Universal Microplate Spectrophotometer. The
cell growth inhibition rate was calculated with the following
equation: % inhibition = [1–(sample group OD490-sample group
OD630)/(control group OD490-control group OD630)] × 100%.
Origin 7.5 software was used to calculate the IC50 values from at
least three independent experiments.
The efficacy of drug combination of FNA with taxol and
camptothecin was evaluated via cell viability assay (MTT assay).
The stock solutions of tested compounds were diluted to the
desired concentrations with culture medium. The cells were
cultured in 96-well plates at a density 5 × 103 cells per well
and incubated until 90–95% confluence, and then 100 µL of
medium containing desired concentrations of test compounds
was added to the wells. Following 48-h incubation, 10 µL of
MTT working solution was added to each well and incubated
for another 4 h. After removal of the medium, 200 µL DMSO
was added to each well to dissolve the formed formazan. The
plates were vortexed for 10 min to ensure complete dissolution.
Then the OD was acquired with a microplate reader at 490 and
630 nm. The cell growth inhibition rate was calculated with the
following equation: % inhibition = [1 – (sample group OD490
– sample group OD630)/(control group OD490 – control group
OD630)] × 100%.
In vivo Antitumor Activity
All animal experiments were performed in compliance with the
Animal Experiment Ethical Review Board of Weifang Medical
University. For in vivo antitumor efficacy studies, male athymic
nude mice (5–6 weeks old, Slac Laboratory Animals, Shanghai,
China) were inoculated subcutaneously in the right shoulder with
1.8 × 107 HepG2 cells.
Injected mice were kept for 10 days; those with palpable
tumors were then randomly assigned into treatment and control
groups (six mice per group). The treatment groups were
administrated with 100 mg/kg/d test compound intragastrically,
whereas the control group was administered with an equal
volume of phosphate-buffered saline (PBS) solution. The tumor
size and body weight were assessed every day. After 15 days of
treatment, the mice were euthanized, and tumor weights were
acquired with an electronic balance. The TGI was calculated
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
ETHICS STATEMENT
using the following formula: TGI = 100% × [1 – (TVt(T)
–
The animal study was reviewed and approved by Weifang
Medical University Ethics Committee.
TVinitial(T))/(TVt(C) – TVinitial(C))], where TVt(T) and TVinitial(T)
stand for the mean tumor volume measured at final time
and at initial time for the treatment groups, respectively, and
TVt(C) and TVinitial(C) represent the mean tumor volume for the
control group.
AUTHOR CONTRIBUTIONS
LZ and WS designed the project. YC synthesized the molecules.
JF performed the enzymatic screening and performed the in
vivo antitumor experiment. YH and XW performed the in
vitro antitumor experiments. YC and LZ analyzed the data and
wrote the manuscript. All authors contributed to the article and
approved the submitted version.
Cell Apoptosis Assay
HepG2 cells in logarithmic growth phase were cultured in 6-
well plates (4 × 105 cells per well).Various doses of FNA and
SAHA (1, 3, and 9 µM) were added and incubated for 24 h.
Then cells were washed with PBS, collected, and resuspended
with binding buffer from a commercially available annexin V–
FITC kit (Thermo Fisher Co., USA) and mixed with 5 µL of
annexin V–FITC gently. Following 10 min of incubation, 1 µL
of propidium iodide was added to the samples and incubated
for another 20 min while avoiding light. Flow cytometry
was used to determine cell apoptosis status (CytoFLEX,
Beckman Coulter).
FUNDING
This work was supported by Science and technology support
plan for youth innovation in universities of Shandong Province
(Grant No. 2019KJM001), Natural Foundation of Shandong
Province (Youth Found, Grant No. ZR2019QH005), National
Natural Science Foundation of China (Youth Found, Grant No.
81803343), International Visiting Scholar Program of Weifang
Medical University (No. 20194-05). The English langue of the
whole manuscript was revised by Qixiao Jiang.
Cell Cycle Analysis
HepG2 cells in logarithmic growth phase were cultured in 6-well
plates with 6 × 105 cells per well and incubated with different
doses of FNA and SAHA (0.125, 0.25, and 0.5 µM). Following
6-h incubation, cells were washed twice with cold PBS and then
fixed in 70% precooled ethanol at 4◦C for 12 h. The fixed cells
were washed again and then stained with PI/RNase A for 30 min
at room temperature.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fonc.
2020.592385/full#supplementary-material
Frontiers in Oncology | www.frontiersin.org
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Nitrogen Mustard HDAC Inhibitor
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