A novel harmine derivative, N-(4-(hydroxycarbamoyl)benzyl)-1-(4- methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide (HBC), as histone deacetylase inhibitor: in vitro antiproliferation, apoptosis induction, cell cycle arrest, and antimetastatic effects

Article abstract of DOI:10.1016/j.ejphar.2018.02.004

This study aims to design and synthesize a novel harmine derivative N-(4-(hydroxycarbamoyl) benzyl)-1-(4-methoxyphenyl)-9H-pyrido [3,4-b]indole-3-carboxamide (HBC) as histone deacetylase (HDAC) inhibitor, and evaluate its antitumor activities and anti-met

Full text of DOI:10.1016/j.ejphar.2018.02.004

Author’s Accepted Manuscript
A
novel
harmine
derivative, N-(4-
(hydroxycarbamoyl)benzyl)-1-(4- methoxyphenyl)-
9H-pyrido[3,4-b]indole-3-carboxamide (HBC), as
histone
deacetylase
inhibitor:
in
vitro
antiproliferation, apoptosis induction, cell cycle
arrest, and antimetastatic effects
www.elsevier.com/locate/ejphar
Jie-Fei Miao, Yan-Fu Peng, Shi Chen, Wei-Jie
Gao, Qiu-Xing Yang, Peng Zhu, Jing Guo, Jinhua
Tao, Lin Luo, Yanan Zhang, Yong Ling
PII:
S0014-2999(18)30084-0
https://doi.org/10.1016/j.ejphar.2018.02.004
EJP71657
DOI:
Reference:
To appear in:
European Journal of Pharmacology
Received date: 28 September 2017
Revised date: 29 January 2018
Accepted date: 7 February 2018
Cite this article as: Jie-Fei Miao, Yan-Fu Peng, Shi Chen, Wei-Jie Gao, Qiu-
Xing Yang, Peng Zhu, Jing Guo, Jinhua Tao, Lin Luo, Yanan Zhang and Yong
Ling, A novel harmine derivative, N-(4-(hydroxycarbamoyl)benzyl)-1-(4-
methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide (HBC), as histone
deacetylase inhibitor: in vitro antiproliferation, apoptosis induction, cell cycle
arrest, and antimetastatic effects, European Journal of Pharmacology,
https://doi.org/10.1016/j.ejphar.2018.02.004
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A novel harmine derivative, N-(4-(hydroxycarbamoyl)benzyl)-1-(4-
methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide (HBC), as histone
deacetylase inhibitor: in vitro antiproliferation, apoptosis induction, cell cycle
arrest, and antimetastatic effects
Jie-Fei Miaoa,b, Yan-Fu Peng b, Shi Chen b, Wei-Jie Gao b, Qiu-Xing Yangb, Peng Zhub, Jing Guob,
Jinhua Tao b, Lin Luo a,b, Yanan Zhang a,b, Yong Ling a,b,c,
aThe Affiliated Hospital of Nantong University, Nantong University, Nantong 226001, PR China;
bSchool of Pharmacy and Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong
University, Nantong 226001, PR China.
cState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, PR China.
Abstract
This study aims to design and synthesize a novel harmine derivative N-(4-(hydroxycarbamoyl)
benzyl)-1-(4-methoxyphenyl)-9H-pyrido [3,4-b]indole-3-carboxamide (HBC) as histone
deacetylase (HDAC) inhibitor, and evaluate its antitumor activities and anti-metastasis mechanism.
HBC not only exerted significant ant-proliferation activity against five human cancer cell lines,
especially for HepG2 cell with an IC50 value of 2.21 μM, which is nearly three-fold lower than
SAHA (IC50 = 6.26 µM), but also showed selective HDAC1/6 inhibitory effects in vitro. However,
HBC had little effect on normal hepatic cells LO2. Furthermore, HBC simultaneously increased
the acetylation of histone H3, H4, and α-tubulin, induced hypochromism by electrostatical
 Corresponding author. Tel.: +86-513-8505-1892; e-mail: lyyy111@sina.com (Y. Ling).
1

interaction with CT-DNA, triggered more significant cancer cell apoptosis and cell cycle arrest at
G2/M than SAHA by inhibition of both CDK1 and cyclin B in a concentration dependent manner.
In addition, scratch and invasion assay showed that HBC also dose-dependently suppressed
migration and invasion capacities of highly metastatic HCC HepG2 cells through down-regulated
the expression of tumor metastasis related proteins MMP-2 and MMP-9, significantly better than
SAHA. Finally, HBC showed low acute toxicity to mice and significant growth inhibition of the
hepatoma tumor in vivo. These results demonstrate that novel harmine-based HDAC inhibitor
HBC not only exhibited selective HDAC1/6 inhibitory activity and significant in vitro and in vivo
antitumor activity, but also possessed DNA binding effect, apoptosis induction, cell cycle arrest
effects, and potent anti-metastasis mechanisms, which may hold great promise as therapeutic
agent targeting HDAC1/6 for the intervention of human cancers.
Keywords: Harmine derivative; Antitumor activity; Histone deacetylase inhibitor; Apoptosis; Cell
cycle arrest; Antimetastatic activity
1. Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignant liver tumors. Most
patients with HCC die within one year after diagnosis largely because of frequent tumor
recurrence and metastasis (Zhao et al., 2016). Surgical resection and liver transplantation are
currently the mainstay of therapy and offer the prospect of long-term survival or even cure;
however, most patients are not suitable because of tumor extent, poor liver dysfunction and
2

limited supply of donor livers. (Johnson, 2005; Raza et al., 2014). Given that most patients HCC
are diagnosed in advanced stages, there is a great clinical need to develop effective, well-tolerated
targeted systemic therapies and chemotherapies.
β-Carbolines are a class of naturally occurring indole alkaloids, with a common tricyclic
pyrido[3,4-b]indole ring structure. Members of this family include harmine, harmaline, harmalol,
and harmane (Fig. 1), which were originally isolated from the seeds of the medical plant Peganum
harmala traditionally used in the North Africa (Cao et al., 2007; De Meester, 1995). These
harmine and its derivatives have been implicated in a wide spectrum of pharmacological activities,
such as intercalation into DNA, inhibition of CDK, topisomerase, DYRK1A, monoamine oxidase,
and antiangiogenesis activity (Cao et al., 2007; Hamsa et al., 2010; Carvalho et al., 2017).
Therefore, harmine and its derivatives have been widely studied as anti-cancer agents. However,
many harmine derivatives show weak anti-cancer effects, which may result from their low tumor
specificity (Nussbaumer et al., 2011).
HDACi’s are inhibitors of the histone deacetylases (HDACs), enzymes that have been found to
be overexpressed in various human cancers and associated with tumorigenesis. Several HDACi’s
have been approved for the treatment of cutaneous T-cell lymphoma (Grant et al., 2010), PTCL or
multiple myeloma (Garnock-Jones, 2015; Lu et al., 2016); however, they generally show a lack of
visible efficacy against solid tumors (Gryder et al., 2012). The combination of harmine derivatives
and histone deacetylase inhibitors may achieve increased anticancer efficacy by simultaneously
targeting multiple pathways. This is particularly important given metastatic cancer cells often
develop resistance against conventional cancer therapies, resulting in greater difficulty to
3

effectively treat malignant cancer (Chen et al., 2013; LaBarge, 2010).
In an effort to augment the therapeutic benefits of these anticancer agents, our group has
undertaken an approach that incorporate the essential structural elements of harmine into HDACi’s.
We recently developed a series of hybrids consisting of the key structural elements of β-carboline
and hydroxamic acid, as present in clinically HDACi’s SAHA, where the phenyl ring was replaced
with a β-carboline aromatic heterocyclic ring as the CAP group in classic HDAC inhibitors,
connected to the zinc-binding group (ZBG) with a linker consisting of alkyl groups. These
compounds displayed increased antitumor potency in vitro, and one of the most potent compounds
LZ-11c (Fig. 2) displayed significant growth inhibition of cancer cells in vivo and low acute
toxicity (Ling et al., 2015). Importantly, the improved anticancer activities of these hybrid
compounds appeared to be related to both HDAC inhibition, DNA damage, as expected with
β-carbolines. Since many potent and selective HDAC inhibitors such as tubastatin A, chidamide
(Fig. 2), have a benzylic linker (Butler et al., 2010; Lu et al., 2016), instead of the alkyl linker in
SAHA, in the usual CAP-linker-ZBG scaffold, we designed a novel harmine derivatives,
N-(4-(hydroxycarbamoyl)benzyl)-1-(4-methoxyphenyl)-9H-pyrido-[3,4-b]indole-3-carboxamide
(HBC), bearing an NH at the amide position on the benzylic linker (Fig. 2). We herein report the
biological evaluation of the novel hydroxamic acid-based harmine derivative HBC, as well as
investigation on its cell cycle arrest and anti-metastasis mechanisms in multiple cancer cell lines.
2. Materials and Methods
2.1. Chemicals and reagents
4

L-tryptophan (Purity ≥ 98%), 4-methoxybenzaldehyde (Purity ≥ 98%), harmine (Purity ≥
98%), Vorinostat (SAHA, Purity ≥ 99%), and methyl 4-(bromomethyl)benzoate (Purity ≥ 98%)
were purchased from Aladdin Technologies Inc. (Shanghai, China). Compounds 2-3 and 5 were
synthesized according to previously reported procedures by our group (Ling et al., 2017).
Dimethyl sulfoxide (DMSO, cell culture grade, Purity ≥ 99%), and methylthiazolyl-
diphenyltetrazolium bromide (MTT, Purity ≥ 99%) were purchased from Sigma Chemical Co. (St.
Louis, MO, USA). All the media, Roswell Park Memorial Institute (RPMI)-1640 medium,
Dulbecco's modified Eagle's medium (DMEM), heat-inactivated fetal bovine serum (FBS),
streptomycin/penicillin solution used for cell culture were purchased from Macgene Co. (Beijing,
China). All the other reagents were purchased from Aladdin Technologies Inc. (Shanghai, China).
2.2. Prepareation and characterization of HBC
1H NMR spectra were recorded with a Bruker Advance 300 MHz spectrometer at 300 K, using
TMS as an internal standard. MS spectra were recorded on a Mariner Mass Spectrum (ESI). High
resolution mass spectra (HRMS) were recorded using an Agilent Technologies LC/MSD TOF. All
compounds were routinely checked by TLC and 1H NMR. TLCs and preparative thin-layer
chromatography were performed on silica gel GF/UV 254, and the chromatograms were
conducted on silica gel (200–300 mesh, Merck) and visualized under UV light at 254 and 365 nm.
All solvents were reagent grade and, when necessary, were purified and dried by standard methods.
Solutions after reactions and extractions were concentrated using a rotary evaporator operating at
a reduced pressure of ca. 20 Torr. Organic solutions were dried over anhydrous sodium sulfate. All
5

compounds were of >95% purity determined by HPLC.
N-(4-(hydroxycarbamoyl)benzyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamid
e (7, HBC): To a solution of 3 (0.38 g, 1.2 mmol), EDCI (0.27 g, 1.44 mmol) and DMAP (17 mg,
0.14 mmol) in 10 ml anhydrous CH2Cl2, was added 5 (0.20 g, 1.2 mmol). The mixture was stirred
at room temperature overnight. Then 20 ml of CH2Cl2 was added and the mixture was washed
with water (30 ml × 3) and brine. The organic phase was dried over anhydrous sodium sulfate,
filtered and evaporated in vacuo, and the crude product was purified by column chromatography
to give 6. Intermediate 6 was then dissolved in 3 ml anhydrous methanol, and poured into a
solution of NH2OK (0.09 g, 4 mmol) in 3 ml of anhydrous methanol. The mixture was stirred for
10-15h and the solvent was evaporated in vacuo. The residue was diluted with saturated NH4Cl
aqueous solution, and then extracted with ethyl acetate (6 ml × 5). The organic layers were
combined, dried over anhydrous Na2SO4 and evaporated. The resulting residue was purified by
column chromatography (eluting with EA followed by 20:1 CHCl3/MeOH followed by 10:1
CHCl3/MeOH) on silica gel to afford 7 as a pale yellow solid in a yield of 67%. Purity = 98.26%
by HPLC. Analytical data for 7: 1H NMR (DMSO-d6, 300 MHz):  10.79 (s, 1H, NH), 9.02 (s, 1H,
NH), 8.03 (m, 1H, Ar-H), 7.93 (d, 2H, J = 6.0 Hz, Ar-H), 7.71 (d, 2H, J = 6.0 Hz, Ar-H), 7.43-7.49
(m, 4H, Ar-H), 7.11 (m, 4H, Ar-H), 6.70 (m, 1H, Ar-H), 4.63 (d, 2H, J = 6.0 Hz, NCH2), 3.83 (s,
3H, OCH3); MS (ESI) m/z = 467 [M+H]+; HRMS (ESI): m/z calcd for C27H23N4O4: 467.1719;
found: 467.1733 [M+H]+.
2.3. Cell lines and cell culture
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Hepatocellular carcinoma cells (HepG2, SMMC-7721, H22, and Huh7), human colon cancer
cells (HCT116), human lung cancer cells (H1299), and human normal hepatocyte cells (LO2)
were obtained from Shanghai Institute of Cell Biology (Shanghai, China). All cells were cultured
in 10% FBS DMEM (Gibco, NY), and were supplemented with 10% (v/v) heat-inactivated fetal
bovine serum, 2 mM glutamine, HBCM HEPES buffer (4-(2-hydro-xyethyl)-1-piperazine
ethanesulfonic acid), 100 U/ml streptomycin and 100 U/ml penicillinat at 37 °C in a humid
atmosphere (5% CO2, 95% air). For subculture, cells were detached with 0.25% trypsin-EDTA
(Ethylenediaminetetra-acetic acid) solution, and 1×106 cells were seeded into new flasks.
2.4. MTT assay
The cells were cultured in 96-well flat-bottom microplates overnight, and incubated in
triplicate with vehicle or different concentrations of test compound for 48 h. During the last 4 h
incubation, 30 μl of tetrazolium dye (MTT) solution (5 mg/ml) was added to each well. The
resulting MTT-formazan crystals were dissolved in 150 μl DMSO, and absorbance was measured
spectrophotometrically at 570 nm using an ELISA plate reader. The inhibition induced by each
tested compound at the indicated concentrations was expressed as a percentage. The concentration
required for 50% inhibition (IC50) was calculated using the software (GraphPadPrism Version
4.03).
2.5. HDAC activity assay.
HDAC activity assays were performed as previously reported (Ling et al., 2015). The HDAC
7

activity was determined using the HDAC fluorimetric activity assay kit (Enzo Life Sciences Inc.)
according to the manufacturer’s instructions. Briefly, HDAC enzyme solution (HDAC1, HDAC2,
HDAC3, HDAC6, or HDAC8) was incubated with test compounds at different concentrations in
the presence of HDAC substrate (Boc-Lys (Ac)-AMC) at 37 °C for 60 min. Then the lysine
developer was added to stop the reaction. After 30 min, the data was recorded in a fluorescence
plate reader with excitation at 355 nm and emission at 460 nm. The HDAC activity was calculated
as a percentage of activity compared with the control group. The concentration required for 50%
inhibition (IC50) was calculated using GraphPad Prism (Version 4.03).
2.6 UV–visible spectroscopy titrations
UV–visible spectroscopy titrations were performed on an ultraviolet spectrophotometer (UV
2500, Shimadzu, Tokyo, Japan) at 25 °C. Stock solutions of 20 μM of CT DNA (calf thymus DNA,
which can form perfect double stranded DNA structure) were prepared in 5mM Tris buffer (60.50
mg Tris, 292.5 mg NaCl, Ph 7.0, with 100 ml Milli Q water). Stock solutions of 20 μM
synthesized derivative was prepared by dissolving them in 1:1000 DMSO/Milli Q water.
UV–visible absorption titrations were done by adding 100 μl CT DNA solution each time to the
quartz cuvette containing about 4 ml active compound solution. Titrations were carried out until
the complex absorption band remains at a fixed wavelength upon five successive additions of CT
DNA. Absorption spectra were recorded from 250 nm to 400 nm.
2.7. Flow cytometry assay for cell apoptosis.
8

HepG2 cells were cultured overnight and incubated in triplicate with different concentrations
of HBC (2.2 and 6.6 μM), SAHA (5.0 μM), harmine (10 μM), or vehicle for 48 h. The cells were
harvested and stained with APC-Annexin V and 7-AAD at room temperature for 15 min. The
percentage of apoptotic cells was determined by flow cytometry (Calibur, BD, USA) analysis. The
APC signal detector (FL4) (Ex = 633 nm; Em = 660 nm) and 7-AAD staining signal detector (FL3)
(Ex = 488 nm; Em =647 nm) were used to detect the cells with the flow cytometer. Ten thousand
cells were counted for three independent experiments. The data were analyzed using WinList 3D
(version7.1) and the histogram was plotted using Excel 2010.
2.8. Flow cytometry assay for cell cycle analysis
HepG2 (2~3×105 cells/well) were seeded into 6-well plates for 48 h, and incubated in triplicate
with HBC (1.1, 2.2, or 4.4 μM), SAHA (5.0 μM) or vehicle (0.1% of DMSO) in 2 ml DMEM
medium supplemented with 10% FBS for 48 h prior to analysis. The harvested cells were fixed in
70% ethanol at 4 C overnight, followed by incubation with RNase A and staining with PI
(Sigma-Aldrich). The Deoxyribonucleic acid (DNA) content was determined by flow cytometry
using a flow cytometer (Beckman Coulter). The distribution of cell cycles was analyzed using the
ModFit LT for Mac software (Verity, Software House, Topsham, ME, USA).
2.9. Western blot assay
The mechanisms of the inhibitory activity of HDAC, cell cycle-related proteins, and MMPs
were determined in the western blot assay. HepG2 cells at 1.5×105/ml were treated with different
9

dosage of HBC, SAHA, or vehicle control (0.1% of DMSO) in 2 ml DMEM medium
supplemented with 10% FBS for 48 h. After harvested and lyzed, the cell lysates (50 μg/lane)
were separated by SDS-PAGE (12% gel) and transferred onto nitrocellulose membranes. After
blocked with 5% fat-free milk, the target proteins were probed with anti-acetyl-histone H3,
anti-acetyl-histone H4, anti-acetyl-α-tubulin, anti-CDK1, anti-cyclin B, anti-MMP2, anti-MMP9,
and anti-β-actin antibodies (Cell Signaling Technology, MA, USA), respectively. The bound
antibodies were detected by HRP-conjugated second antibodies and visualized using the enhanced
chemiluminescent reagent. The relative levels of each signaling event to control β-actin were
determined by densimetric scanning.
2.10. Wound-healing Assay
HepG2 cells were seeded in a 96-well tissue culture plate at 1.5×104 cells/well and incubated
overnight in the presence of 10 % FBS. Then, the center of the cell monolayer was scratched with
a sterile micropipette tip to create a scratch of constant width. Subsequently, cellular debris was
washed with PBS, and HepG2 cells were exposed to various concentrations of HBC and SAHA.
Wound closure was monitored and photographed at 0 and 24 h with an Olympus IX51.
2.11. Transwell invasion assays
Invasion assays were performed following the manufacturer’s instructions (BD Biosciences).
Cells were treated at time of plating. After 24 h (as noted in the legend), invasion cells were fixed
to the Matrigel_(BD Biosciences)-coated transwell membranes with a seeding density of 5×104
10

cells per well for 48 h and stained with crystal violet (0.1 % in 20 % methanol), and invaded cells
visualized and quantified by microscopy. Data represented as number of invaded cells per field of
view ± S.D. for triplicate experiments.
2.12. Antitumor effects of HBC in mice.
H22 cells (3 × 106) were subcutaneously injected into the right armpit of the mice that were
randomly divided into four groups of six mice each. Treatments were initiated when tumors
reached a mean group size of approximately 100 mm3. The groups with HBC treatment received
two dosages (43 or 86 μmol/kg) by intravenous injection. The negative control group received
0.9% normal saline, and the positive group was treated with SAHA by intraperitoneal injection (86
μmol/kg). The corresponding agent for each group was administered every day for 19 days. All
mice were then sacrificed, and their tumors were segregated and weighed. The tumor inhibitory
ratio was calculated by the following formula: tumor inhibitory ratio (%) = [(Wcontrol
−
Wtreated)/Wcontrol] × 100%. Wtreated and Wcontrol were the average tumor weights of the treated and
control mice, respectively. The tumor diameters were measured with calipers every three days, and
the tumor volume was calculated by the formula V (mm3) = d2 × D/2, where D is the largest
diameter and d the smallest diameter.
2.13. Statistical analysis
Data obtained from different experiments were presented as means ± S.D. (standard of
deviation) from at least three independent experiments and evaluated by Student-Newman-Keuls
11

test. Values of P < 0.05 were considered statistically significant.
3. Results
3.1. Chemistry
The synthetic route of compound HBC is outlined in Scheme 1. Treatment of commercially
available L-tryptophan 1 with a solution of 4-methoxyphenaldehyde in HOAc produced the
intermediate 2 via a Pictet-Spengler reaction, which was then oxidized by KMnO4 in DMF to
furnish compound 3. Methyl 4-(bromomethyl)benzoate 4 was treated with ammonia aqueous in
THF to obtain 5. Reaction of 3 with 5 in the presence of 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDCI) and 4-dimethylamino- pyridine (DMAP) afforded amide 6.
Finally, treatment of intermediate 6 with NH2OK gave the target compound 7 (HBC). HBC,
purified by column chromatography, was of >95% purity determined by high-performance liquid
chromatography (HPLC), and its structure was confirmed by 1H NMR, MS, and HRMS.
3.2. In vitro antitumor activity and selectivity.
HBC was first tested for its cancer cell growth inhibitory activity against hepatocellular
carcinoma cells (HepG2, SMMC-7721, H22, and Huh7), human colon cancer cells (HCT116), and
human lung cancer cells (H1299) in MTT assays. SAHA and harmine, a natural β-carboline
analogue with anti-tumor activity, were used as control. The IC50 values of HBC against five
human cancer cell lines are listed in Table 1. Compound HBC displayed good antiproliferative
activity with IC50’s in the low micromole range in these cells, which were comparable or lower
12

than that of SAHA, and also significantly lower than harmine. In particular, the IC50 values of
HBC were 2.21 and 2.32 µM in HepG2 and HCT116 cells, respectively, which were two-to-three
times lower than those of SAHA (IC50 = 6.26 and 5.53 µM), and 20 times lower than those of
harmine (IC50 = 51.2 and 46.7 µM).
Given the significant growth inhibitory activity of HBC in vitro, the selectivity profile was
investigated by examining the inhibitory effects of HBC on the growth of HepG2 and LO2 cells. It
was found that treatment with increased dose of HBC had no significant effect on the survival of
non-tumor LO2 cells, whereas the same treatment induced marked inhibition of HepG2 cells
proliferation (Fig. 3). These results suggest that HBC possesses selectivity in antiproliferation
activity against tumor cells over normal cells.
3.3. In vitro HDAC inhibitory activity of HBC.
Encouraged by the antiproliferative activity of HBC against five human cancer cell lines, we
further tested HBC for its in vitro inhibitory activities against several HDAC enzymes. SAHA was
used as the positive control. The potencies of HBC were obtained by measuring the
fluorescent-based HDAC biochemical activity using recombinant human HDAC1, HDAC2,
HDAC3, HDAC4, HDAC6, and HDAC8 enzymes and the IC50 values are summarized in Table 2.
Compound HBC had high inhibitory potencies against HDAC1 and HDAC6 with IC50 values of
4.1 ± 0.6 nM and 2.6 ± 0.3 nM respectively, which were nearly 20 to 30-fold lower than HDAC3,
and much lower than HDAC2, HDAC4, and HDAC8. The IC50 values of HBC was 25-fold lower
13

than SAHA on HDAC1, and was 22-fold lower than SAHA on HDAC6.
3.4. HBC induces acetylation of histone H3, H4, and α-tubulin.
To further investigate whether HBC induces acetylation of histones at the cellular level, we
analyzed the expression of the acetylation status of histone H3, H4, and α-tubulin in western
blotting assays, using β-actin as a loading control (Fig. 4). HepG2 cells were incubated with the
vehicle alone, SAHA (5.0 μM), or HBC (1.1, 2.2, and 4.4 μM) for 48h. We found that HBC not
only had significant effect on histone H3 and H4 acetylation, but also increased the expression of
acetyl-α-tubulin in a dose-dependent manner. Levels of acetyl-histone H3, acetyl-histone H4, and
acetyl-α-tubulin in most of the HBC treated groups (2.2 and 4.4 μM) were higher than the 5.0 µM
SAHA treated group, which was consistent with the results from the HDAC isoform fluorimetric
activity assay.
3.5. HBC induces DNA binding effect
It is known that harmine, with the planar structured β-carbolines, can bind to DNA and induce
DNA damage (Vignoni et al., 2014). To investigate whether the anti-cancer activities of HBC
resulted partly from harmine with DNA–ligand binding effect, UV-visible spectroscopic titration
studies were performed. In general, hypochromism (reduction in absorption) is observed when a
small molecule binds to DNA through intercalation, resulting from a strong π–π stacking
interaction between an aromatic chromophore from the molecule and the base pairs of DNA
(Kamal et al., 2015). In our study, the HBC-CT-DNA system showed absorption bands at 300 nm
14

and 355 nm, with the maximum absorption at 300 nm (Fig. 5). Upon addition of equal increments
of CT-DNA to HBC solution, the intensity of all absorption bands gradually decreased, with the
strongest hypochromicity observed at 355 nm. A similar trend was observed in harmine, with the
maximum absorption at 320 nm. However, the absorption slightly increased with SAHA upon
addition of incremental CT-DNA (see support information). These results clearly indicated
electrostatic binding of HBC to CT-DNA with intercalation.
3.6. HBC induces tumor cell apoptosis
To determine whether the inhibitory effects of HBC on hepatocellular carcinoma cell
proliferation were accompanied by enhanced cancer cell apoptosis, HepG2 cells were incubated
with different concentrations of HBC, SAHA, or harmine for 48 h, and the percentages of
apoptotic cells were determined by FITC-Annexin V/PI staining and flow cytometry. As shown in
Fig. 6, the percentages of annexin V + apoptotic HepG2 cells gradually increased for those cells
exposed to the increasing concentrations of HBC (41.33% for 2.2 μM and 71.02% for 6.6 μM),
which were better than the positive control groups SAHA (35.7% for 5.0 μM) and harmine (24.5%
for 10 μM). These results demonstrated that the incubation with HBC induced HepG2 cell
apoptosis in a dose-dependent manner.
3.7. HBC induces G2/M cell cycle arrest
We next investigated whether the significant anti-proliferative activity of HBC resulted from
the induction of cell cycle arrest. We analyzed the cellular DNA content using flow cytometric
analysis in propidium iodide (PI) stained cells to detect changes in the cell cycle distribution.
15

Fig. 7A and B demonstrated the cell cycle profile of HepG2 cells after incubation with different
concentrations of HBC or SAHA for 48 h. Compared to the control cells treated with DMSO,
HBC led to significant induction of G2/M phase cell cycle arrest in a concentration-dependent
manner, which was consistent with harmine-treated cell cycle arrest effect (Song et al., 2004). This
is evident from the increasing percentage of cells in peak corresponding to G2/M phase,
accompanied by a proportionate reduction in cells in other phases of the cell cycle (Fig. 7A). The
percentages of G2/M phase fraction at 2.2 and 4.4 μM of HBC was significantly higher than that
of SAHA at 5.0 μM.
3.8. HBC regulated the expression of G2/M-related proteins
G2/M transition is largely dependent on cyclin B1 and CDK1 activity which play important roles in
the regulation of the cell cycle (Roskoski, 2016; Höckner et al., 2016). To further explore the
underlying mechanisms of G2/M phase arrest induced by HBC, we examined the inhibitory effects of
HBC on the expression of cyclin B1/CDK1. HepG2 cells were incubated with the vehicle alone, SAHA
(5.0 μM), or HBC (1.1, 2.2, and 4.4 μM) for 48h. The expression levels of the cyclin B1 and CDK1
were determined by immunoblotting assays using β-actin as the loading control. As shown in Fig. 7C,
treatment with HBC caused a drastic decrease in the expression of cyclin B and CDK1 in HepG2 cells,
which was similar to harmine reported in the literature (Song et al., 2004). These results clearly
demonstrate that HBC potentially induce G2/M phase cell cycle arrest in HepG2 cells, which might be
responsible for their antiproliferative effect.
16

3.9. HBC Suppress Metastatic Activities of HCC Cells in Vitro
Cell invasion and migration are highly relevant properties in the process of tumor metastasis
(Singh et al., 2014). To investigate whether a novel HDAC inhibitor HBC affects the process of
HCC cell metastasis and invasion, we studied HBC for cell invasion and migration, using SAHA
as a positive control. In the wound-healing migration assay, cell line monolayer of HCC cells
(HepG2) was scratched and the percentage of wound closure was monitored. HepG2 cell were
incubated with DMSO (control group), HBC (1.0 and 2.0 μM), or SAHA (2.0 μM) for 24 h. As
shown in Fig. 8, HBC was effective in arresting cell motility. The migration rate of HBC (1.0 and
2.0 μM) treated cells was over 50% lower than the control group. The migration rate of HBC (2.0
μM) treated cells was also significantly lower than SAHA (2.0 µM) treated group, suggesting that
HBC effectively inhibited cell migration with greater potency than SAHA.
The transwell assay was also used to determine the impact of HBC on HepG2 cell migration
and invasion. Transwell insets were precoated with basement membrane matrix, and the number
of HepG2 that penetrated the membrane was quantified. We found that HBC inhibited HepG2 cell
metastasis in a dose-dependent manner. As shown in Fig. 9, treatment with HBC significantly
decreased the number of invaded cells with the percentage of 28.8% ± 3.09% (P < 0.01) at
concentrations of 2.0 μM, which was much lower than that of SAHA 40.5% ± 5.67%, (P < 0.01)
at the same concentration. Therefore, these results clearly showed that HBC significantly inhibited
cell migration and invasion in a concentration-dependent manner.
3.10. HBC inhibits the expression of MMP-2 and MMP-9
17

Matrix metalloproteinases (MMPs) also play key roles in tumor invasion and metastasis, and
over-expression of MMPs such as MMP-2 and MMP-9 have been well correlated with cell
invasion and migration in many cancers (Khan et al., 2015; Salo et al., 1994). In this experiment,
western blotting was performed to investigate the effects of HBC on the regulation of the
expression of MMP-2 and MMP-9 in HepG2 cells. Compared with the control group, the
expression of MMP-2 was dose-dependently down-regulated with the treatment with HBC (Fig.
10). The expression of MMP-9 also had slightly decrease after the treatment with HBC. These
results suggest that HBC decrease the cell migration and invasion by decreasing the expression of
matrix metalloproteinases-2 and -9.
3.11. In vivo anti-cancer activity of HBC
To evaluate the safety of HBC in vivo, groups of ICR mice were injected intraperitoneally with
a single dose of HBC or vehicle control, respectively. The survival of mice was monitored up to
14 days after injection. Only one mice that had been treated with HBC at the highest dose (610.4
mg/kg) survived (shown in Table 3). In contrast, injection with HBC at the lowest dose (250
mg/kg) did not cause any death and abnormality in eating, drinking, body weight, and activity
throughout the observation period. As a result, the LD50 value of HBC was calculated to be 463.3
mg/kg for this train of mice.
Next, we established a mice model which was inoculated subcutaneously with H22 cells to
evaluate the in vivo antitumor activity of HBC. After the establishment of solid tumor, the mice
were randomly intraperitoneally administered with HBC, SAHA, and vehicle, respectively. The
18

changes in tumor volumes and weights were measured over 19 days. Compared with the vehicle
control, treatment with HBC significantly reduced the tumor volume growth rate in a
dose-dependent manner (Fig. 11). It was observed that treatment with HBC (86 μmol/kg) strongly
inhibited the growth of the tumor cells in vivo similar to SAHA at the same dose. Importantly, the
tumor weights (0.87 ± 0.12, 0.61 ± 0.08 g) in the mice treated with HBC at 43 and 86 μmol/kg,
respectively, were significantly less than those from the vehicle-treated controls (1.58 ± 0.23 g, P
< 0.01). Besides, there was no statistical difference in body weight in postinoculation among the
four groups of mice. Together, our data demonstrated that HBC could evidently inhibit the growth
of tumor in vivo.
4. Discussion
Many plant-derived agents with few adverse effects have been developed as potential
pharmacotherapies for HCC. Harmine, a tricyclic pyrido[3,4-b]indole ring alkaloid, has been
implicated in a variety of pharmacological activities related to cancer, such as intercalation into
DNA, inhibition of CDK (Liu et al., 2016; Song et al., 2004; Sharma et al., 2016), cell cycle
progression, cell apoptosis and autophagy (Cao et al., 2011; Li et al., 2017; Vignoni et al., 2014;
Daoud et al., 2014).
In the current study, we described the design and syntheis of a novel hydroxamic acid-based
harmine derivative HBC which incorporated the key structural components of harmine and
classical HDACi’s, and evaluated its in vitro biological activities in an extensive battery of assays.
19

Data obtained from in vitro HDAC inhibition assay showed that HBC is selective for HDAC1 and
HDAC6. Compared with SAHA, compound HBC is 22- to 25-fold more potent against HDAC1
and HDAC6. Furthermore, compound HBC suppressed proliferation in six cancer cell lines with
IC50 ranging from 2.21 μM to 4.32 μM. However, HBC had little effect on normal hepatic cells
LO2 (Fig. 2), suggesting that HBC possesses selectivity in antiproliferation activity against tumor
cells over normal cells. In addition, HBC also dose-dependently induced tumor cell apoptosis and
cell cycle arrest in G2/M period in HepG2 cells, which was similar to harmine (Figures 6 and 7).
Finally, a low concentration of HBC decreased the cell migration and invasion, possibly by
reducing the expression of matrix metalloproteinases-2 and -9. These results all demonstrated that
HBC was potent in inhibiting HepG2 cell proliferation and metastasis in vitro.
Global hypo-acetylation of histone H4 has been reported as a common hallmark of human
tumors occurring early in the tumorigenic process (Grolla et al., 2009). The anticancer effect of
HDAC inhibitors could be also assessed by their potency in inducing hyper-acetylation of histone
H3 or H4 (Fraga et al., 2005). It is known that histone H3 is a substrate for HDACs 1, 2, and 3,
and α-tubulin is a substrate for HDAC6 (Khochbin et al., 2001). In this paper, the efficacy of HBC
on tumor cell acetylated histone and α-tubulin levels was evaluated by Western blot. Notably,
compound HBC significantly upregulated the level of acetylated histone H3, H4 and α-tubulin in a
dose-dependent manner (Fig. 3). These results are in agreement with our current data that HBC
selectively inhibited HDAC1 and HDAC6 activity, which may lead to fewer side effects than
pan-HDAC inhibitors.
20

Control of cell cycle progression is considered an effective strategy for tumor treatment. It has
been reported that harmine induces cell cycle arrest in diverse cancer cells (Liu et al., 2016; Song
et al., 2004). The ability to cause mitotic arrest reportedly correlates with higher cytotoxicity of
HDAC inhibitors (Blagosklonny et al., 2002). Our data showed that harmine derivative HBC
treatment for 48 hours induced G2/M phase arrest of cell cycle progression (Fig. 7), and decreased
the expression of CDK1 and cyclin B in HepG2 cells. These results are consistence with harmine
reported in the literature (Song et al., 2004), and indicate that the cell cycle arrest is one of the
mechanisms of inhibition of HepG2 cell proliferation.
Metastasis has been found to be accompanied by various physiological alterations, which
contribute to the degradation of epithelial-mesenchymal transition (EMT), as well as invasion and
migration of tumor cells via the blood stream or lymphatic system. This also includes subsequent
transport of tumor cells to other tissues and organs (Fidler, 2003). EMT is a critical process in
tumor progression that promotes cell breaching of the basement membrane and metastasis to
distant sites. Characteristic changes associated with EMT enable the migration and invasion of
cancer cells. Deregulated cell or cell-matrix adhesion and increased cell migration are
preconditions for the progression of metastatic cancer. MMPs have a crucial role in the cancer cell
migration and invasion (Khan et al., 2015; Zou et al., 2016). In particular, MMP-2 and MMP-9 are
known to degrade the ECM in a process associated with cancer progression, invasion, and
metastasis. Thus, many studies have focused on the levels of MMPs which are involved in the
21

metastasis process (Eissa et al., 2007). Based on these observations, we investigated the effects of
HBC on the invasive ability of HepG2 cells. Our experiments showed that HBC could suppress
the migratory and invasive ability by inhibiting the activities and expression of MMP-2 and
MMP-9 in HepG2 cells (Fig. 10).
Our study demonstrated that the antimetastatic effect of harmine derivative HBC as an
HDAC1/6 inhibitor against the metastatic HCC cell line HepG2 in vitro. HBC inhibited the
metastasis of HCC cells by blocking cell cycle and suppressing metastatic phenotypes such as
migration and invasion. These results suggest that HBC may serve as an effective therapeutic
agent for hepatic metastasis treatment. Further studies on HBC are necessary to investigate its
antimetastatic effects and mechanisms in various cancer cell lines and clinical cases.
5. Conclusion
In the paper, we report a novel hydroxamic acid-based harmine derivative HBC which
incorporated the key structural components of harmine and classical HDACi’s, and evaluate its in
vitro biological activities in an extensive battery of assays. HBC selectively inhibits tumour cell
proliferation but not normal cell LO2, and exerts significant HDAC1/6 inhibition. Furthermore,
treatment with HBC effectively causes accumulation of both acetylated histones and acetylated
α-tubulin, and promotes cell apoptosis in HepG2 cells. In addition, HBC induces cell cycle arrest
via enhanced the expression of CDK1 and cyclin B in HepG2 cells. HBC also suppresses
migration and invasion capacities of highly metastatic HCC HepG2 cells through down-regulated
22

the expression of tumour metastasis related proteins MMP-2 and MMP-9 in a concentration
dependent manner. Finally, HBC displayed low acute toxicity and significant growth inhibition of
the hepatoma tumor in vivo. Together, given their potent antitumor activities, these 3-substituted
harmine-based HDACi’s warrant further investigation as candidates for the potential treatment of
human cancer.
Authors’ contributions
Yong Ling and Jie-Fei Miao designed the study. Jie-Fei Miao and Shi Chen synthesized
harmine derivative HBC. Wei-Jie Gao, Jing Guo, and Qiu-Xing Yang carried out the experiments.
Lin Luo and Yan-Fu Peng analysed the data. Yanan Zhang, Yong Ling, and Jie-Fei Miao wrote
the paper. All authors have read and approved the manuscript.
Declaration of interest
The authors declare no conflict of interest and are solely responsible for the content of this
manuscript.
Acknowledgements
We gratefully acknowledge the financial support by the Natural Science Foundation of China
(Grant Nos. 81302628 and 81473089), the Project of “Jiangsu Six Peaks of Talent”
(2014-SWYY-044 and 2016-SWYY-CXTD-008), China Pharmaceutical University for the Open
Project Program of State Key Laboratory of Natural Medicines (SKLNMKF201611), China and
23

Jiangsu Province Postdoctoral Science Foundation (2016M590488 and 1601136B), the Project of
“Jiangsu 333 high-level talents”, Jiangsu Government Scholarship for Overseas Studies
(JS-2014-212), Applied Research Projects of Nantong City (MS12015060), and also thank a
project funded by the Priority Academic Programs Development of Jiangsu Higher Education
Institutions (PAPD).
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Fig. 1. The structure of harmine and its analogs harmaline, harmalol, and harmane.
Fig. 2. Structures of clinically approved HDAC inhibitors and the design of β-carboline-based
hydroxamic acid derivative HBC.
Fig. 3. Inhibitory effects of HBC on the proliferation of HepG2 and LO2 cells. Cells were
incubated with the indicated concentrations of HBC for 48 h. Cell proliferation was assessed using
the MTT assay. Data are means ± S.D. of the inhibition (%) from three independent experiments.
Fig. 4. Immunoblot analysis of histone H3 and H4 acetylation and α-tubulin acetylation in vitro.
(A) HepG2 cells were treated with compounds HBC and SAHA for 48 h at the indicated
concentrations using western blot analysis. Cell lysates were prepared and subjected to
SDS-PAGE and immunoblot analysis using anti-acetyl- histone H3, anti-acetyl- histone H4,
anti-acetyl-α-tubulin, and anti-β-actin antibodies, respectively. β-Actin serves as a control for
protein loading. (B) Quantitative analysis. The relative levels of Ac-H3, Ac-H4, and Ac-α-tubulin
used to control β-actin were determined by densimetric scanning. The data are expressed as means
± S.D. of three separate experiments. *P< 0.01 vs control.
Fig. 5. UV-visible absorption spectra of HBC in the presence of increasing amounts of CT DNA.
Arrows indicate the changes in absorbance with increasing the concentration of DNA. [HBC] = 20
μM.
Fig. 6. Compound HBC induces HepG2 cell apoptosis in vitro. HepG2 cells were incubated with
the indicated concentrations of HBC, SAHA, or harmine for 48 h, and the cells were stained with
FITC-Annexin V/PI, followed by flow cytometry analysis. (A) Flow cytometry analysis. (B)
Quantitative analysis of apoptotic cells. Data are expressed as means ± S.D. of the percentages of
29