ZYH005, a novel DNA intercalator, overcomes all-trans retinoic acid resistance in acute promyelocytic leukemia

Article abstract of DOI:10.1093/nar/gky202

Despite All-trans retinoic acid (ATRA) has transformed acute promyelocytic leukemia (APL) from the most fatal to the most curable hematological cancer, there remains a clinical challenge that many high-risk APL patients who fail to achieve a complete molecular remission or relapse and become resistant to ATRA. Herein, we report that 5-(4-methoxyphenethyl)-[1, 3] dioxolo [4, 5-j] phenanthridin-6(5H)-one (ZYH005) exhibits specific anticancer effects on APL and ATRA-resistant APL in vitro and vivo, while shows negligible cytotoxic effect on non-cancerous cell lines and peripheral blood mononuclear cells from healthy donors. Using single-molecule magnetic tweezers and molecule docking, we demonstrate that ZYH005 is a DNA intercalator. Further mechanistic studies show that ZYH005 triggers DNA damage, and caspase-dependent degradation of the PML-RARa fusion protein. As a result, APL and ATRA-resistant APL cells underwent apoptosis upon ZYH005 treatment and this apoptosis-inducing effect is even stronger than that of arsenic trioxide and anticancer agents including 5-fluorouracil, cisplatin and doxorubicin. Moreover, ZYH005 represses leukemia development in vivo and prolongs the survival of both APL and ATRA-resistant APL mice. To our knowledge, ZYH005 is the first synthetic phenanthridinone derivative, which functions as a DNA intercalator and can serve as a potential candidate drug for APL, particularly for ATRA-resistant APL.

Full text of DOI:10.1093/nar/gky202

Nucleic Acids Research, 2018
1
doi: 10.1093/nar/gky202
ZYH005, a novel DNA intercalator, overcomes all-trans
retinoic acid resistance in acute promyelocytic
leukemia
Qingyi Tong1,†, Huijuan You1,†, Xintao Chen1, Kongchao Wang1, Weiguang Sun1,
Yufeng Pei1, Xiaodan Zhao2, Ming Yuan3, Hucheng Zhu1, Zengwei Luo1,* and
Yonghui Zhang1,*
1Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical
College, Huazhong University of Science and Technology, Wuhan 430030, China, 2Mechanobiology Institute,
National University of Singapore, 117411, Singapore and 3School of Pharmacy, Hubei University of Chinese
Medicine, Wuhan 430065, China
Received January 30, 2018; Revised March 02, 2018; Editorial Decision March 07, 2018; Accepted March 09, 2018
ABSTRACT
can serve as a potential candidate drug for APL, par-
ticularly for ATRA-resistant APL.
Despite All-trans retinoic acid (ATRA) has trans-
formed acute promyelocytic leukemia (APL) from
the most fatal to the most curable hematologi-
cal cancer, there remains a clinical challenge that
many high-risk APL patients who fail to achieve
a complete molecular remission or relapse and
become resistant to ATRA. Herein, we report
that 5-(4-methoxyphenethyl)-[1, 3] dioxolo [4, 5-
j] phenanthridin-6(5H)–one (ZYH005) exhibits spe-
cific anticancer effects on APL and ATRA-resistant
APL in vitro and vivo, while shows negligible
cytotoxic effect on non-cancerous cell lines and
peripheral blood mononuclear cells from healthy
donors. Using single-molecule magnetic tweezers
and molecule docking, we demonstrate that ZYH005
is a DNA intercalator. Further mechanistic stud-
ies show that ZYH005 triggers DNA damage, and
caspase-dependent degradation of the PML-RARa
fusion protein. As a result, APL and ATRA-resistant
APL cells underwent apoptosis upon ZYH005 treat-
ment and this apoptosis-inducing effect is even
stronger than that of arsenic trioxide and anticancer
agents including 5-fluorouracil, cisplatin and doxoru-
bicin. Moreover, ZYH005 represses leukemia devel-
opment in vivo and prolongs the survival of both
APL and ATRA-resistant APL mice. To our knowl-
edge, ZYH005 is the first synthetic phenanthridinone
derivative, which functions as a DNA intercalator and
INTRODUCTION
Normally, cells are equipped with DNA damage response
(DDR) pathways and damage to DNA is detected and re-
paired. However, most cancer cells have relaxed DDR path-
ways, and more importantly, they are capable of ignoring
DNA damage and allowing cells to achieve high prolifer-
ation rates, increasing their susceptibility to DNA damage
drugs compared to that of normal cells since replication of
damaged DNA increases the possibility of cell death (1,2).
Consequently, the concept of targeting DNA in cancer ther-
apy has inspired the development of numerous anticancer
drugs, particularly DNA-binding drugs such as cisplatin,
carboplatin, oxaliplatin, mitoxantrone, amsacrine, temo-
zolomide and anthracyclines (3–5). Despite dose-limiting
side effects, the extensive use of these DNA-binding drugs in
clinical practice has revealed their utility, and they will con-
tinue to be a staple in anticancer regimens. Meanwhile, the
discovery of new DNA-binding drugs with improved effects
and a high specificity for cancer cells is of great importance.
DNA-binding drugs include covalent binding ligands
(alkylating agents) and non-covalent ligands (groove
binders and intercalators) (5). DNA intercalators, which
bind DNA by inserting aromatic moieties between adja-
cent DNA base pairs, have attracted considerable atten-
tion due to their potent anticancer activity. For exam-
ple, several acridine and anthraquinone derivatives (i.e. an-
thracycline) are excellent DNA intercalators that are cur-
rently available on the market and widely used as anti-
cancer agents (6,7). Acridine and anthraquinone represent
*To whom correspondence should be addressed. Tel: +86 27 83692868; Fax: +86 27 83692762; Email: zhangyh@mails.tjmu.edu.cn
Correspondence may also be addressed to Zengwei Luo. Email: luozengwei@hust.edu.cn
†The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.
ꢀ
C
The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.
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2 Nucleic Acids Research, 2018
two of the main frameworks of DNA intercalators, and
the other well-known framework is phenanthridine (6). For
many decades, phenanthridine derivatives have been recog-
nized for their efficient DNA intercalative binding capabil-
ity (8) and have been applied as gold-standard DNA/RNA-
fluorescent markers (ethidium bromide, EB) and probes for
DNA (propidium iodide, PI); however, they are also consid-
ered disadvantageous due to their potential genotoxic and
mutagenic effects. In the past decade, Amaryllidaceae alka-
loids with a phenanthridinone rather than phenanthridine
skeleton, such as narciclasine, cis-dihydronarciclasine, 7-
deoxypancratistatin, lycoricidine and pancratistatine, have
been reported to have potent and selective anticancer ef-
fects (9–12). Importantly, narciclasine and other natural
phenanthridinone alkaloids are considered potentially use-
ful drug leads for the treatment of apoptosis-resistant can-
cers and metastatic cancers (13–15). In addition, synthetic
phenanthridinone derivatives which exhibited greater an-
ticancer activity than several standard chemotherapeutics
(Taxol, doxorubicin, gemcitabine and cisplatin) also have
been reported (16). And some phenanthridinone deriva-
tives have been proven to be potent PARP inhibitors
(6(5H)-phenanthridinone, PJ34) (17,18), topoisomerase I
inhibitors (ARC-111 and its analogues) (19,20), all of which
exhibit pronounced and targeted antitumor activity. There-
fore, phenanthridinone alkaloids and their derivatives have
been under intense scrutiny and are currently being pursued
as clinical candidates for cancer treatment (12,14,21,22).
These findings prompted us to develop novel phenanthridi-
none derivatives with selective anticancer effects and to ex-
plore whether these derivatives act as DNA intercalators in
the same manner as phenanthridines.
Acute promyelocytic leukemia (APL) is the M3 sub-
type of acute myeloid leukemia (AML), with 98% of pa-
tients harboring the t(15;17) chromosomal translocation,
involving the fusion of the genes encoding PML (promye-
locytic leukemia) and RAR␣ (retinoic acid receptor alpha)
(23–26). In addition, impaired homologous recombination
(HR) (25,27,28), non-homologous end-joining (NHEJ) re-
pair (25) and base excision repair (BER) (27) pathways have
been found in APL, all of which are considered essential
contributors to APL pathogenesis. All-trans retinoic acid
(ATRA) and arsenic trioxide (ATO) are PML-RAR␣ tar-
geting drugs that bind to the RAR␣ and PML moieties,
respectively. These two drugs have transformed APL from
the most fatal to the most curable hematological cancer
(23,29,30). Despite the unprecedented success, many high-
risk patients fail to achieve complete molecular remission
or relapse and become resistant to ATRA (31,32). There-
fore, alternative agents must be developed, particularly for
relapsed APL with ATRA resistance.
kaloids is hampered by a lack of efficient supply, since to-
tal syntheses are complex and biotechnological approaches
are completely missing. In this study, we synthesized 20
phenanthridinone-based alkaloids using this method and
found that ZYH005 exerts specific anticancer effects on
APL and ATRA-resistant APL models. Mechanistically,
ZYH005 exerts its effects through intercalative binding to
DNA, which subsequently triggers DNA double-strand
breaks and the accumulation of DNA damage, causes
G2/M cell cycle arrest and caspase-dependent degradation
of PML-RARa. As a result, APL and ATRA-resistant APL
cells underwent apoptosis upon ZYH005 treatment. No-
tably, this apoptosis-inducing effect of ZYH005 was found
to be stronger than that of ATO and widely used anticancer
agents (5-fluorouracil, cisplatin and doxorubicin). To the
best of our knowledge, this is the first report of a synthesized
phenanthridinone alkaloid that functions as a DNA inter-
calator and has potential therapeutic value for the treatment
of both APL and relapsed APL with ATRA resistance.
MATERIALS AND METHODS
Synthesis of ZYH005 and its analogues
Briefly, the phenanthridinone was synthesized from a ke-
toacid through transition metal-free decarboxylative cy-
clization (38). Then, the appropriate side chain was in-
troduced to obtain ZYH005 through a nucleophilic sub-
stitution reaction. The compounds ZYH001-ZYH004 and
ZYH006-ZYH020 were obtained using the same procedure
as that used to synthesize ZYH005, with different starting
materials. A detailed description of the process by which
these compounds were synthesized, as well as their 1H
NMR data (13C NMR data for selected compounds), is in-
cluded in the Supplementary Information.
Reagents
All-trans retinoic acid, nitroblue tetrazolium (NBT) and
dimethylsulfoxide (DMSO) were purchased from Sigma-
Aldrich (MO, USA). Arsenic trioxide was obtained from
Tongji Hospital (Wuhan, China). Cremophor® EL was
purchased from Aladdin Chemicals (Shanghai, China). The
cell lysis buffer, BCA Protein Assay Kit and ACK buffer
were purchased from Beyotime Biotechnology (Shang-
hai, China). Matrigel was purchased from BD Biosciences
(CA, USA). The Wright-Giemsa Staining Kit was pur-
chased from Jiancheng Bioengineering Institute (Nanjing,
China). Z-VAD-FMK, 5-fluorouracil and cisplatin were
purchased from Selleck Chemicals (Shanghai, China). Dox-
orubicin, idarubicin, chloroquine and MG-132 were pur-
chased from MedChemExpress (NJ, USA). Antibodies
against PARP, cleaved-caspase-3, Bcl-2, Bax, Bak, ␥H2AX,
␤-actin, GAPDH and the appropriate secondary antibod-
ies were purchased from Cell Signaling Technology (MA,
USA). Polyclonal antibodies against RAR␣ (C-20, sc-551)
and PKC␦ (C20, sc-937) were purchased from Santa Cruz
Biotechnology (CA, USA).
We previously isolated and identified 24 new Amaryl-
lidaceae alkaloids (33–35), and demonstrated that N-
methylhemeanthidine chloride, which with a phenanthri-
dine core, exhibited prominent anticancer effects on pan-
creatic cancer and AML in vitro and in vivo (36,37). Later,
we developed an environmentally friendly and inexpensive
procedure in which the phenanthridinone skeleton was syn-
thesized through Na2S2O8-promoted decarboxylative cy-
clization of biaryl-2-oxamic acid (38). This is of great sig-
nificance because the development of phenanthridinone al-
Mice
BALB/c-nu/nu mice were purchased from Beijing HFK
Bioscience Co., Ltd. (Beijing, China), and FVB/N mice
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Nucleic Acids Research, 2018 3
Single-molecule magnetic tweezers assay
were purchased from Beijing Vital River Laboratory Ani-
mal Technology Co., Ltd. (Beijing, China). The mice were
housed under specific pathogen-free (SPF) conditions and
handled in accordance with the Guidelines for the Care and
Use of Laboratory Animals of Tongji Medical College of
Huazhong University of Science and Technology.
The 6618 bp dsDNA (∼50% GC) was prepared by PCR us-
ing TaKaRa Ex Taq DNA Polymerase (TaKaRa, Dalian,
China) on a lambda phage DNA template (Thermo Sci-
entific, MA, USA ). The primers (Sangon Biotech, Shang-
hai, China) were labeled with 5ꢁ-biotin and 5ꢁ-thiol, respec-
tively. The DNA was tethered between a streptavidin-coated
paramagnetic bead (Dynabeads M-280, Thermo Scien-
tific, MA, USA) and a (3-Aminopropyl) triethoxy silane
(APTES, Sigma-Aldrich, MO, USA) -functionalized cover-
slip, through a biotin-streptavidin interaction and covalent
bond (sulfo-SMCC crosslinker, Thermo Scientific, MA,
USA). The height of the bead above the coverslip sur-
face was measured based on the bead image using single-
molecule magnetic tweezers (43). Extension changes in ds-
DNA were measured based on force-jump measurements
using single-molecule magnetic tweezers (43). At each force,
the magnet was held for 5 s to measure extension changes
at equilibrium from the average bead height.
The torsional constraint DNA for the supercoiling as-
say was prepared by ligating the 6573 bp DNA with a
510 bp dsDNA handle containing ∼50 biotinylated dUTP
(Biotin-11-dUTP, Thermo Scientific, MA, USA) at one end
and a 510 bp handle containing ∼50 digoxigenin-dUTP
(Digoxigenin-11-dUTP, Roche, Basel, Switzerland) as re-
ported in a previous study (44). dsDNA was tethered be-
tween a streptavidin coated 1-␮m diameter paramagnetic
bead (Dynabeads MyOne, Thermo Scientific, MA, USA)
and an anti-digoxigenin antibody (Thermo Scientific, MA,
USA) coated coverslip. The rotation of the magnetic bead
was controlled by rotating a permanent magnet pair using
a rotary motor as reported in a previous study (44). The
rotation-extension curve data were recorded by measuring
DNA extension for 20 s at different magnet turns.
Cell lines and cell culture
The APL cell lines NB4, NB4-MR2 (39), NB4-LR2
(40), hPML-RAR␣ and mutant hPML-RAR␣ transgenic
mouse-derived leukemic cells (41) were kind gifts from Pro-
fessor Guoqiang Chen and Yingli Wu (Shanghai Jiao Tong
University, Shanghai, China). The DND-41, KOPT-K1 and
CUTLL1 cell lines were kindly provided by Dr Warren Pear
(University of Pennsylvania, USA) and Professor Hudan
Liu (Wuhan University, China) and maintained as previ-
ously described (42). The MOLT4, K562, Kg1a, THP-1,
Kasumi, and HL60 cell lines were purchased from Ameri-
can Type Culture Collection (VA, USA). HPDE6-C7 and
NCM460 cell lines were purchased from the Institute of
Biochemistry and Cell Biology of the Chinese Academy of
Sciences (Shanghai, China). Cells were incubated at 37◦C
in a humidified atmosphere of 5% CO2/95% air (v/v).The
leukemia cells were cultured in RPMI 1640 medium (Hy-
clone, UT, USA), and the other cells were cultured in Dul-
becco’s modified Eagle’s medium (Hyclone, UT, USA), both
types of media were supplemented with 10% (v/v) heat-
inactivated FBS. All cell lines were cultured for fewer than 6
months after resuscitation and tested for mycoplasma con-
tamination using the MycoAlert Mycoplasma Detection kit
(Lonza, Slough, UK).
Cell viability assay
Cell viability was measured with an MTS Kit (Promega,
WI, USA). Briefly, the cells were seeded into 96-well plates
at a density of 5000 cells/well and incubated with or with-
out drugs. After 24 h, 20 ␮l of MTS was added to the wells.
The cells were subsequently incubated in the dark for 3 h;
then, the optical density values were measured at 490 nm.
The 50% inhibitory concentration (IC50) for each drug was
calculated using the SPSS software.
Molecular docking
Compounds were subjected to molecular docking experi-
ments using ICM 3.8.1 modeling software (45) on an Intel i7
4960 processor (MolSoft LLC, CA, USA). Molecules were
built with Chemdraw and optimized at molecular mechan-
ical and semiempirical levels using Open Babel GUI. The
X-ray crystallographic structure of the DNA dodecamer d
(CGTACG)2 with doxorubicin was selected from the Pro-
tein Data Bank (PDB code: 2GB9) for the docking study
(46). Ligand-binding pocket residues were selected using
graphical tools in the ICM software to create the bound-
aries of the docking search. In the docking calculation, po-
tential energy maps of the receptor were calculated using
default parameters. Compounds were imported into ICM
and an index file was created. Conformational sampling was
based on the Monte Carlo procedure, and finally the lowest-
energy and the most favorable orientation of the ligand were
selected.
Isolation and culture of peripheral blood mononuclear cells
(PBMCs)
Experiments involving healthy volunteers (a healthy 31-
year-old male, a healthy 31-year-old female, and a healthy
27-year-old male) donated blood were conducted with prior
approval of Research Ethics Board of the Huazhong Uni-
versity of Science and Technology, with informed consent
obtained from all subjects. Peripheral blood mononuclear
cells (PBMCs) from healthy volunteers 1, 2 and 3 (PBMCs-
V1, PBMCs-V2, PBMCs-V3) were collected and isolated by
density gradient centrifugation (12). The isolated PBMCs
were maintained in RPMI 1640 media supplemented and
maintained in the same way as the APL cell lines. PBMCs
were treated within 1 h of collection with various doses of
ZYH005 for up to 24 h; the cell viability was measured with
an MTS Kit.
Immunofluorescence assay
Cells treated with or without ZYH005 were placed on slides
and fixed in ice-cold 4% paraformaldehyde for 15 min. Af-
ter permeabilization with 0.1% (v/v) Triton X-100 in PBS
and blocking with 2% (w/v) bovine serum albumin (BSA)
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4 Nucleic Acids Research, 2018
in PBS, the cells were incubated with an antibody against
␥H2AX overnight at 4◦C. Then, the cells were stained
with the appropriate secondary antibody for 1 h, mount-
ing medium with DAPI (Invitrogen, MA, USA) was added
to the slides, and cover slips were mounted onto the slides
in a manner that did not create bubbles. Fluorescence sig-
nals were detected with an LSM710 confocal microscope
(ZEISS, Weimar, Germany). For the quantitative analysis,
␥H2AX foci were counted by eye during the microscopic
examination and imaging process using a 100× objective.
At least 100 cells per group were examined, and three inde-
pendent experiments were performed.
RESULTS
Selection of ZYH005 for subsequent experiments
Alkaloids with N-phenylethyl phenanthridinone exhibited
more potent cytotoxic activity (33). Therefore, we syn-
thesized compounds with methoxyl, benzyl, phenylethyl,
phenylpropyl and (4-methoxylphenyl) ethyl substituents at
the hetero nitrogen atom of the phenanthridinone ring
(ZYH001-ZYH005) (Supplemental Figure S1A). We pre-
liminarily assessed their anti-proliferation effects on five
cancer cell lines (HL60, SMMC-7721, A549, MCF-7,
SW480), and found that ZYH005 inhibits the proliferation
of all cancer cell lines at low concentrations after 48 h of
treatment, especially the proliferation of the AML cell line
HL60 (IC50 = 0.037 ␮M). Moreover, ZYH005 was more
effective than the other N-alkyl derivatives and the positive
control cisplatin (DDP) (Supplemental Figure S1B). Then,
we focused on N-(4-methoxylphenyl) ethyl derivatives and
synthesized compounds ZYH006–ZYH020 (Supplemental
Figure S2A). Further investigation of the anti-proliferation
effects of ZYH005–ZYH020 on HL60 cells showed that
compounds ZYH006–ZYH020 exerted weaker inhibitory
effects than ZYH005 (Supplemental Figure S2B). There-
fore, ZYH005 was selected for subsequent pharmaceutical
research experiments (Figure 1A, Supplementary methods).
Cell cycle, apoptosis assay and immunoblot analysis
Cell cycle, apoptosis assay and immunoblot analysis were
done as previously describe d (47,48).
Xenograft tumor mouse model assay
A total of 5 × 106 NB4 cells in a PBS/Matrigel solu-
tion (1:1) were subcutaneously injected into the flanks of
5–6-week-old BALB/c-nu/nu mice. After the cells formed
palpable tumors (100–150 mm3), the mice were randomly
assigned to groups treated with vehicle (5% DMSO, 5%
Cremophor® EL, 90% Saline) or ZYH005 (10 mg/kg/day,
intravenously) according to the preliminary toxicity test.
Two weeks later, the animals were sacrificed; their tumors
were removed, weighed and photographed. The visceral or-
gans and tumors were collected and fixed in 10% forma-
lin for hematoxylin and eosin (H&E) or immunohistochem-
istry (IHC) analysis.
1
The H spectral and 13C NMR spectral data for ZYH005
are shown in Supplemental Figure S3.
ZYH005 treatment selectively inhibits the proliferation of
APL and ATRA-resistant APL cells
To explore the anti-leukemia potential of ZYH005, we
treated ten leukemia cell lines and two immortalized nor-
mal human epithelial cell lines with ZYH005 (0–0.16 ␮M)
and then assessed their viability. As shown in Figure 1B,
even after treatment for only 24 h, ZYH005 exerted sig-
nificantly greater anti-proliferation effects on NB4 and
HL60 cell lines than on the other cell lines. Furthermore,
ZYH005 exerted minimal effects on the viability of the nor-
mal cell lines NCM460 and HPDE6-C7. The 24 h IC50
values for the NB4 and HL60 cell lines were 0.041 and
0.053 ␮M, respectively. We further assessed the effects of
ZYH005 on ATRA-resistant cell lines. After a 24 h of treat-
ment, high ATRA concentrations (12.5–50 ␮M) had al-
most no effect on the proliferation of the NB4-LR2 and
NB4-MR2 cell lines. In contrast, ZYH005 at the concentra-
tions of 0.04–0.06 ␮M inhibited the proliferation of these
cell lines (Figure 1C). The effects ZYH005 on peripheral
blood mononuclear cells isolated from blood samples of 3
healthy volunteers (PBMCs-V1, PBMCs-V2, PBMCs-V3)
were also detected. Interestingly, the viability of PBMCs in
the ZYH005-treated groups was nearly consistent with that
in the non-treated groups, even at a ZYH005 concentra-
tion up to 0.64 ␮M (Figure 1D). Induction of promyelocytic
leukemic cell differentiation plays an important role in the
response of APL to both ATRA and ATO treatments (24).
Therefore, we evaluated the differentiation-inducing effects
of ZYH005. However, unlike ATRA and ATO, ZYH005
did not induce APL cell differentiation (Supplemental Fig-
ure S4A and B). These data demonstrate that ZYH005 is a
novel molecule that is selectively cytotoxic to both APL and
Transplantation of ATRA-sensitive/-resistant leukemic mice
and treatment
The female FVB/N mice (6–7 weeks old) were sublethally
irradiated (4.5 Gy), then 2 × 105 viable leukemic cells
isolated from spleens of leukemic hPML-RAR␣ or mu-
tant hPML-RAR␣ transgenic mice were intravenously in-
jected via tail vein (41,49,50). Four days after transplanta-
tion, the mice were randomly assigned to groups treated
with vehicle (5% DMSO, 5% Cremophor® EL, 90%
saline) or ZYH005 (10 mg/kg/day, intravenously). Normal
mice (irradiated but not transplanted, no treatment) and
mice treated with ATO (5 mg/kg/day, intraperitoneally)
or ATRA (15 mg/kg/day, intraperitoneally, for ATRA-
resistant APL model) were used as the controls. Cytological
and histological analyses were performed as reported (41).
Statistical analysis
Comparisons between groups were performed using a stan-
dard two-tailed Student’s t-test or one-way analysis of vari-
ance (ANOVA) followed by Tukey’s post hoc test and stated
in the figure legends. All experiments were repeated at least
three times. The data are presented as the mean S.D., and
P values < 0.05 were considered significant.
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Nucleic Acids Research, 2018 5
Figure 1. ZYH005 treatment specifically inhibits the proliferation of APL and ATRA-resistant APL cells. (A) The synthesis process of 5-(4-
methoxyphenethyl)-[1, 3] dioxolo [4,5-j] phenanthridin-6(5H)–one (ZYH005). (B–D) The viability of cell lines was evaluated using an MTS kit after treated
with drugs for 24 h. The effects of ZYH005 on two immortalized normal human epithelial cell lines (NCM460 and HPDE6-C7) and ten leukemia cell lines
are shown in B. The effects of ZYH005 and ATRA on ATRA-resistant cell lines are shown in C. Effects of ZYH005 on peripheral blood mononuclear
cells isolated from blood samples of 3 healthy volunteers (PBMCs-V1, PBMCs-V2 and PBMCs-V3) are shown in D. Data are shown as the mean S.D.
of three independent experiments, unpaired two-tailed student’s test was used for statistics, **P < 0.01 compared to the control group (DMSO < 0.1%).
ATRA-resistant APL cell lines, has a lesser effect on other
leukemia cell lines, and has a negligible cytotoxic effect on
non-cancerous cell lines as well as PBMCs from healthy
donors.
or the so-called B-to-S overstretching transition (53). At
a low concentration of ZYH005 (1 ␮M), the overstretch-
ing transition force increased, suggesting that the binding
of ZYH005 stabilizes B-form DNA. Increased overstretch-
ing transition forces and stabilization of B-form DNA have
also been observed with other DNA intercalators such as
EB (52). Taken together, ligand-induced dsDNA elonga-
tion and overstretching force alteration at a low concen-
tration and ligand-induced dsDNA extension both suggest
that ZYH005 binds to dsDNA as an intercalator.
ZYH005 intercalates double-stranded DNA
To determine whether ZYH005 could directly interact with
double-stranded DNA (dsDNA), single-molecule magnetic
tweezers were firstly used to investigate the effects of
ZYH005 on dsDNA mechanical properties. The binding of
small ligands to dsDNA alters its structure, causing length-
ening or unwinding of the dsDNA (51,52). Figure 2A shows
the single-molecule stretching experiment system for mea-
suring the force-extension curves of a 6618 bp dsDNA teth-
ered between a coverslip and a paramagnetic bead, as de-
tailed in the methods section. Force was applied to the DNA
molecule through the bead by a pair of magnets. Extension
changes were measured based on the bead height using the
diffraction pattern of the bead image. Figure 2B shows typi-
cal force-height curves of a dsDNA molecule in the presence
of different concentrations of ZYH005 in buffer change ex-
periments. The force-height curves of dsDNA showed sig-
nificant elongation from 20 to 60 pN after adding ZYH005,
which was concentration-dependent, suggesting that the
binding of ZYH005 increased the length of dsDNA as is
typically observed for DNA intercalators (51,52). In addi-
tion to lengthening dsDNA, the binding of ZYH005 also
changed the behavior of the DNA overstretching transi-
tion. The naked dsDNA showed sudden elongation (Fig-
ure 2B, black curve) at a force of ∼65 pN, representing the
transition of B-form DNA to S-DNA (stretched DNA),
To quantify the binding affinity of ZYH005 to dsDNA,
titration curves of dsDNA elongation at different forces
were fit using Hill equation (Figure 2C), xc − xnacked DNA
=
cn
(xsaturate − xnacked DNA
)
, where the xc − xnacked DNA
Kdn(F)+cn
is the elongation of dsDNA at different ligand concen-
tration, xsatureate − xnacked DNA is saturated dsDNA elonga-
tion, Kd(F) is the dissociation constant of ZYH005 with
DNA at different forces, and n = 1 is the Hill efficiency.
The dissociation constant for intercalation of ZYH005 with
DNA at zero force Kd0 is estimated to be 24 ␮M (binding
constant Ka = 1/Kd0 = 4.1 × 104 M−1) by fitting the force-
dependent binding constant (Figure 2D) with the exponen-
tial function, Kd(F) = Kd0 × e−Fꢀx/k T, where kB is the
B
Boltzmann constant and T is temperature. The fitting pa-
rameter ꢀx = 0.1 nm indicates the dsDNA elongation upon
a single ZYH005 molecule intercalation.
To further analyze the effects of ZYH005 on supercoiled
dsDNA and to determine whether the binding of ZYH005
causes dsDNA unwinding, we performed a supercoiling as-
say using torsional constraint dsDNA and magnetic tweez-
ers (Figure 2E). The preparation of torsional constraint ds-
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6 Nucleic Acids Research, 2018
Figure 2. ZYH005 intercalates double-stranded DNA. (A) Binding of ZYH005 with dsDNA measured based on dsDNA stretching experiments. Schematic
diagram of DNA stretching with magnetic tweezers. A 6618 bp dsDNA was tethered between a 2.8 ␮m-diameter paramagnetic bead and coverslip. (B)
Typical DNA stretching curves in the presence of various ZYH005 concentrations. (C) Titration curves of dsDNA elongation measured at different forces
were fit using the Hill equation. The elongation was measured from three independent measurements. (D) Dissociation constant dependent on force. (E)
Effect of ZYH005 on DNA rotation-extension curves at low force (0.4 and 1.0 pN) and saturated ZYH005 concentration (100 ␮M). The 6573 bp DNA
exhibits torsional constraint when rotation = 0; DNA is in relaxed conformation. The inserts depict the states of super coiled DNA under different tensional
and torsional constraints. (F) Low-energy binding conformation of ZYH005 bound to DNA fragments generated by virtual ligand docking.
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Nucleic Acids Research, 2018 7
DNA and twisting of DNA by magnetic tweezers is de-
tailed in the methods section. In the presence of a saturating
concentration of ZYH005 (100 ␮M), evident elongation of
DNA extension at 0.6 pN with negative rotations indicates
that binding of ZYH005 causes DNA stiffen at negative su-
percoiled conformation, suggesting that ZYH005 binds to
dsDNA at low- force (∼0.6 pN) regions. The shift of the
buckling transition at 1 pN also indicates that ZYH005 di-
rectly interacts with dsDNA. Compared to other intercala-
tive compounds such as YOYO-1 and EB, the peak of the
supercoil curve did not show a significant shift, indicating
that ZYH005 binds to dsDNA, but it did not induce signif-
icant unwinding of the dsDNA.
Finally, molecular docking simulations were carried out
to elucidate the interaction of ZYH005 with DNA frag-
ments under default conditions. ZYH005 was expected to
show significantly higher binding affinity with a significant
ICM score (–30.11). Furthermore, the lowest-energy bind-
ing conformation is shown in Figure 2F; the docked pose
was stabilized electronically by ␲–␲ stacking between the
aromatic group and the side chains of the DNA base pairs.
The results obtained from molecular docking studies were
consistent with the results of DNA binding studies, indicat-
ing that ZYH005 is a DNA intercalator.
to induce apoptosis in NB4 cells, and 0.05 ␮M ZYH005
could induce 57.7% and 49.9% (on average) of the cells un-
derwent apoptosis in NB4 and HL60 cells, respectively (Fig-
ure 4A, Supplemental Figure S5B). Next, we evaluated the
effects of ZYH005 on the expression of marker proteins of
apoptosis (59). As shown in Figure 4B, ZYH005 treatment
for 24 h altered the expression levels of Bcl-2, Bax, Bak,
cleaved-caspase-3 and PARP in a dose-dependent manner.
In addition, treatment with 0.05 ␮M ZYH005 could al-
ter the expression levels of cleaved-caspase-3 and PARP
within 6 h, indicating that ZYH005-induced apoptosis is
also a time-dependent phenomenon (Figure 4C). We also
found that ZYH005 treatment induced proteolytic cleavage
of PKC␦, a cytoplasmic caspase-3 substrate, into a 41kDa
catalytic fragment in a time- and dose-dependent manner
(Supplemental Figure S5C). Moreover, 0.05 ␮M ZYH005
induced apoptosis in ATRA-resistant APL cell lines after 24
h of treatment, whereas treatment with a 200-times greater
concentration of ATRA (10 ␮M) did not result in apoptosis
induction (Figure 4D). Meanwhile, the immunoblot analy-
sis showed that ZYH005 treatment altered the expression
of apoptosis-related proteins in ATRA-resistant APL cell
lines (Figure 4E). We compared the apoptosis-inducing ac-
tivity of ZYH005 with that of ATO and other chemother-
apeutics in NB4 and NB4-LR2 cells. As shown in Figure
4F, ATO, 5-fluorouracil, cisplatin and doxorubicin did not
exhibit apoptosis-inducing activity at the indicated concen-
tration and time (0.05 ␮M, 24 h). In contrast, the apop-
tosis induction effect of ZYH005 was observed close to
that of idarubicin, the most effective anthracyclines in both
in vitro assay (60) and in clinical trials (61). These results
strongly support ZYH005 as an effective apoptosis inducer
that induces apoptotic cell death in both APL and ATRA-
resistant APL cell lines.
ZYH005 treatment induces DNA damage and cell cycle ar-
rest in APL and ATRA-resistant APL cells
DNA intercalators with anticancer effects lead to structural
changes in DNA and subsequently cause DNA damage
(54,55). Double-stranded DNA breaks (DSBs) are proba-
bly the most detrimental of the many types of DNA dam-
age occurring within the cell (56). Accordingly, we detected
␥H2AX foci, which are considered indicative of an early re-
sponse to DSBs (57). As shown in Figure 3A and B, NB4
and HL60 cells displayed significant levels of ␥H2AX foci
after 0.05 ␮M ZYH005 treatment for 6 h. By immunoblot
analysis, we found that ZYH005 treatment increased the
levels of ␥H2AX in both APL and ATRA-resistant APL
cell lines (Figure 3C). We further observed that ZYH005
treatment increased the expression levels of ␥H2AX in a
time-dependent manner (Figure 3D). The cell cycle is a crit-
ical regulator of the processes of cell proliferation and divi-
sion after DNA damage occurs. High levels of damage to
DNA induce cell-cycle arrest to prevent the transmission of
damaged DNA during mitosis. Therefore, we detected the
cell cycle distribution in cell lines treated with or without
low-dose ZYH005 for 24 h and found that ZYH005 induced
a significant G2/M cycle arrest in both APL and ATRA-
resistant APL cells (Figure 3E, Supplemental Figure S5A).
ZYH005 treatment induces caspase-dependent PML-RAR␣
degradation
The PML-RAR␣ fusion protein determines not only the
phenotype of APL but also the response of APL to treat-
ment (23), and several studies have shown that PML-
RARa expression induces strong resistance to apoptosis
(62). Therefore, we analyzed the effects of ZYH005 on
PML-RAR␣. As shown in Figure 5A, the PML-RAR␣
levels were significantly decreased in NB4, NB4-LR2 and
NB4-MR2 cells after ZYH005 treatment for 24 h. Caspase
family members have been recognized as key participants in
apoptosis and play important roles in PML-RAR␣ degra-
dation (63,64). Therefore, we treated cells with ZYH005 in
the presence or absence of the caspase inhibitor Z-VAD-
FMK. As shown in Figure 5B, in both APL and ATRA-
resistant APL cells, the ZYH005-induced apoptosis was
significantly attenuated by the addition of Z-VAD-FMK.
Additionally, the immunoblot results showed that Z-VAD-
FMK blocked ZYH005-induced PML-RAR␣ degradation
and alterations of apoptosis-related proteins such as Bcl-2,
Bax and cleaved-caspase-3; however, proteins related to the
DNA damage response, such as PARP and ␥H2AX, were
partially blocked by Z-VAD-FMK (Figure 5C), indicating
that ZYH005-induced DNA damage is an event that occurs
earlier than PML-RAR␣ degradation and apoptosis.
ZYH005 treatment induces apoptotic cell death in APL and
ATRA-resistant APL cells
Damaged DNA accumulation and cell cycle arrest are acti-
vators of apoptotic signals (58), we also noted cell shrink-
age and chromatin condensation in APL cells treated with
ZYH005, and these findings prompted us to investigate
whether ZYH005 could induce apoptosis in these cells.
Therefore, we quantified the apoptosis cells by flow cytome-
ter. With 24 h treatment, ZYH005 at 0.025 ␮M is sufficient
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8 Nucleic Acids Research, 2018
Figure 3. ZYH005 treatment induces DNA damage and cell cycle arrest in APL and ATRA-resistant APL cell lines. (A) Immunofluorescence microscopy
analysis of ␥H2AX foci in NB4 and HL60 cells after 0.05 ␮M ZYH005 treated for 6 h. (B) Quantification of the percentage of cells with ≥ 6 ␥H2AX foci.
At least 100 cells per group were examined. (C, D) Immunoblot analysis of ␥H2AX expression in cell lines after treated with ZYH005 at the indicated
concentrations and times. (E) ZYH005 induces G2/M arrest in APL cells. After treated with DMSO (<0.1%) and ZYH005 for 24 h, cells were harvested,
fixed and cell cycle distribution was analyzed by PI staining. (A, C and D), the data are representative of three independent experiments. (B and E) the
data are expressed as the mean S.D. of three independent experiments, **P < 0.01, ***P < 0.001 unpaired two-tailed Student’s t-test. ␤-Actin was used
as a loading control in immunoblot analysis.
A lysosome inhibitor (chloroquine), proteasome in-
hibitor (MG132) and RNF4 (the E3 ubiquitin ligase of
PML) shRNA–silencing NB4 cells (65,66) were used to de-
termine whether the autophagy-lysosome and proteasome-
ubiquitin pathways had roles in the effect of ZYH005 on
PML-RAR␣ degradation, respectively. The results showed
that all of them failed to attenuate the apoptosis and
PML-RAR␣ degradation induced by ZYH005 (Supple-
mentary Figure S6A–E). Moreover, Z-VAD-FMK could
block apoptosis and PML-RAR␣ degradation in RNF4
shRNA-transfected NB4 cells (Supplementary Figure S6F
and G). These results suggest that ZYH005-induced DNA
damage activates caspase 3 and then induces caspase-
dependent PML-RARa degradation and apoptosis.
tal Figure S7A). Expression of Ki67, which is an impor-
tant marker of cell proliferation (67), was significantly lower
in tumor tissues following ZYH005 treatment (Supplemen-
tal Figure S7B). Moreover, ZYH005 injection did not af-
fect the body weights of the tested animals (Supplemental
Figure S7C). The H&E staining of visceral organs includ-
ing heart, liver, spleen, lung and kidney were also relatively
constant between ZYH005-treated mice and vehicle-treated
mice (Supplementary Figure S7D), manifesting minimal
toxicity of ZYH005 in vivo, which is the precondition to be
a clinical candidate agent.
Next, we transplanted leukemic cells isolated from
spleens of leukemic hPML-RAR␣ or mutant hPML-
RAR␣ transgenic mice intravenously into sub-lethally ir-
radiated isogenic FVB/N recipients to generate APL mice
or ATRA-resistant APL mice (41,49,50). On day 4 after
transplantation, mice were treated intravenously with vehi-
cle, ZYH005, ATO (for APL mice) or ATRA (for ATRA-
resistant APL mice). All mice were sacrificed for histologi-
cal analyses when the first vehicle-treated mouse was mori-
bund. In both APL and ATRA-resistant APL leukemic
mouse models, results of Wright-Giemsa staining showed
that mice in ZYH005-treated group had near-normal pe-
ripheral blood (PB), and leukemic blasts were rarely ob-
served in their bone marrow (BM); results of histologi-
ZYH005 treatment induces leukemia regression in APL and
ATRA-resistant APL mice
To explore the anti-leukemia effect of ZYH005 in vivo,
we first established NB4 xenograft mouse models and
treated the mice with vehicle or ZYH005 (intravenously, 10
mg/kg/day according to the preliminary test). Two weeks
later, we noted significant differences in the tumor weights
between the ZYH005- and vehicle-treated mice, with a tu-
mor inhibition rate of 62.3% (P = 0.0048) (Supplemen-
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Nucleic Acids Research, 2018 9
Figure 4. ZYH005 treatment induces apoptotic cell death in APL and ATRA-resistant APL cell lines. (A) NB4 and HL60 cells were treated with ZYH005
for 24 h, and then cell apoptosis was determined. (B) Immunoblot analysis of the expression of apoptosis-related proteins in NB4 and HL60 cells after 24 h
of ZYH005 treatment. (C) Immunoblot analysis of the expression of PARP and cleaved-caspase-3 in NB4 and HL60 cells after 0.05 ␮M ZYH005 treated
for different time. (D) ATRA-resistant cells were treated with 0.05 ␮M ZYH005 and 10 ␮M ATRA for 24 h, and then cell apoptosis was determined.
(E) Immunoblot analysis of apoptosis-related proteins in ATRA-resistant cells after ZYH005 treated for 24 h. (F) NB4 and NB4-LR2 cells were treated
with ZYH005, arsenic trioxide (ATO), 5-fluorouracil (5-Fu), cisplatin (DDP), doxorubicin (DOX) and idarubicin (IDA) at 0.05 ␮M for 24 h, and then
cell apoptosis was determined. (A, D and F), for DOX and IDA treatment, cell apoptosis was determined by an Annexin V-APC/7-AAD staining kit;
for other treatments, cell apoptosis was determined by an Annexin V-FITC/PI staining kit. Data are expressed as the mean S.D. of three independent
experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant compared to ZYH005-treated group; unpaired two-tailed Student’s t-test in A and
D; one-way ANOVA analysis followed by Tukey’s post hoc test in F.␤-Actin was used as a loading control in immunoblot analysis.
cal examination with H&E staining revealed that leukemic
cell infiltration into spleens and livers was inhibited by
ZYH005 (Figure 6A and B). Moreover, vehicle-treated mice
exhibited enlarged spleens and livers, which could be allevi-
ated by ZYH005 treatment (Figure 6C and Supplementary
Figure S8A). ZYH005 was also found to facilitate recov-
ery of swollen spleens and livers, even in ATRA-resistant
leukemic mice (Figure 6D and Supplementary Figure S8B).
Finally, the effect of ZYH005 on the survival of leukemic
mice was also evaluated using the same treatment proce-
dure as described above but using at least six mice for each
treatment group. All vehicle-treated leukemic mice devel-
oped leukemia within 24–28 days post-transplantation and
died within the following 4 days. When the first vehicle-
treated mouse died, we stop the treatments. Both APL and
ATRA-resistant APL mice showed longer survival follow-
ing treatment with ZYH005 (Figure 6E and F). These re-
sults indicate that ZYH005 has significant therapeutic effi-
cacy against APL and ATRA-resistant APL in vivo.
Myeloid cells positive for four markers (CD34+, c-kit+,
Fc␥RIII/II+, Gr1int) in APL mice have been identified as
leukemia initiating cells (LIC), and PML-RARa degrada-
tion triggers LIC clearance (68,69). Therefore, we analyzed
these cells in APL mice and found that the numbers of these
cells were decreased in the BM of ZYH005-treated mice, re-
sult similar those noted in ATO-treated mice (Supplemen-
tary Figure S8C), indicating that ZYH005 can eliminate
LIC in leukemic mice, which mirrors PML-RAR␣ degrada-
tion in vivo. In addition, we conducted immunohistochemi-
cal analysis of cleaved caspase-3 expression in the spleens
of APL and ATRA-resistant APL mice, and the results
showed that ZYH005 treatment induced apoptosis in the
APL mice similarly to ATO treatment (70) (Supplementary
Figure S8D). We also performed immunoblot analyses us-
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10 Nucleic Acids Research, 2018
Figure 5. ZYH005 induces caspase-dependent PML-RAR␣ degradation and apoptosis. (A) Immunoblot analysis of PML-RAR␣ expression in NB4 and
ATRA-resistant cells treated with ZYH005 for 24 h. (B and C) NB4 and ATRA-resistant cell lines were treated with 80 ␮M Z-VAD-FMK for 1 h, and
then 0.05 ␮M ZYH005 was added. 24 h later, cell apoptosis was determined by an Annexin V-FITC and PI staining kit (B). Data are expressed as the
mean S.D. of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed Student’s t-test. The expression levels of related
proteins were evaluated by immunoblot analysis (C). GAPDH was used as a loading control.
ing NB4 xenograft tumor lysate, and the results showed that
ZYH005 treatment activated caspase-3 and induced PML-
RAR␣ degradation in vivo (Supplementary Figure S8E).
Collectively, these data provide evidence of the mech-
anisms of ZYH005 treatment, which mainly mediate in-
tercalative binding to DNA, subsequently trigger DNA
double-strand breaks and accumulation of DNA damage,
induce G2/M cell cycle arrest, caspases 3 activation and ul-
timately cause PML-RAR␣ degradation and apoptosis in
both APL and ATRA-resistant APL models (Figure 6G).
been used to characterize the binding mode of drug-DNA
interactions (51), the binding affinity (52), binding kinet-
ics (71) and the effects of drugs on the supercoiled dsDNA
(72). Using single-molecule magnetic tweezers, we show
that ZYH005-binding increases DNA extension and twists
DNA, which suggesting that ZYH005 is a DNA intercala-
tor. It should be noted that ZYH005 is a moderate inter-
calator when comparing with classical intercalators such as
YOYO-1 and EB, as ZYH005 only causes moderate change
of the extension and twists of DNA at low force. We further
demonstrate that ZYH005 exerts its effects by triggering
DNA damage, cell cycle arrest, caspase-dependent degrada-
tion of PML-RARa and apoptosis. These findings highlight
the potential of ZYH005 for APL therapy and for overcom-
ing ATRA resistance.
DISCUSSION
In this study, we report that ZYH005, a novel synthe-
sized phenanthridinone alkaloid, has specific and effective
cytotoxic effects in APL and ATRA-resistant APL mod-
els. In contrast, ZYH005 showed negligible toxic effect on
PBMCs from healthy volunteers as well as non-cancerous
cells. Single-molecule force-spectroscopy experiments have
Cells respond to DNA damage through DDR signalling
networks, which determine cell fate and promote not only
DNA repair and cell survival but also cell cycle arrest and
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Nucleic Acids Research, 2018 11
Figure 6. Effects of ZYH005 on APL and ATRA-resistant APL mouse models. On day 4 after transplantation, APL and ATRA-resistant APL mice
were treated with vehicle (5% DMSO, 5% Cremophor® EL, 90% Saline) or ZYH005 (10 mg/kg/day, intravenously). Normal mice (irradiated but not
transplanted, no treatment) and mice treated with ATO (5 mg/kg/day, intraperitoneally) or ATRA (15 mg/kg/day, intraperitoneally, for the ATRA-
resistant APL model) were used as the controls. (A–D) When the first vehicle-treated leukemic mice were moribund, all mice were sacrificed and analyzed.
Peripheral blood (PB) and bone marrow (BM) were subjected to Wright-Giemsa staining. Invasion of the spleen and liver by leukemic cells was detected
by hematoxylin and eosin (H&E) staining, the arrowheads indicate infiltrating cells (A and B). The spleens and livers were weighed (C and D), *P < 0.05,
**P < 0.01, ***P < 0.001, unpaired two-tailed Student’s t-test. (E–F) Kaplan–Meier survival curves of APL (E) and ATRA-resistant APL (F) mice. The
numbers of mice are indicated in parentheses, and ***P < 0.001 compared to the vehicle-treated mice. Every experiment was repeated at least three times
with the similar results. (G) Diagram of signaling pathways regulated by ZYH005 in APL and ATRA-resistant APL.
cell death, thus determining the outcome of cancer ther-
apy with DNA-binding drugs (1). Therefore, defects in the
DDR have been exploited therapeutically in the treatment
of cancer with DNA-binding chemotherapies and PARP in-
hibitors (2,73). Compromised DDR has been found in APL
(25,27,28), indicating that APL cells may be sensitive to
DNA-binding drugs. Associated with this, before the intro-
duction of ATRA and ATO for APL, the first-line therapy
for APL was DNA intercalators (daunorubicin, doxoru-
bicin, idarubicin) plus the DNA synthesis inhibitor cytara-
bine (Ara-C), and the complete remission rate was as high
as 75% (23,74). Until now, ATRA in combination with these
DNA binding chemotherapies remained standard practice
for APL treatment (23,31,74). Recently, Esposito et al. re-
ported that APL is extremely sensitive to DDR target drugs
(PARP inhibitors) due to their compromised DDR (28).
These findings suggest that the impairment DDR not only
determines APL pathogenesis but also plays an important
role in the therapy response of APL. Moreover, molecu-
lar mechanism underlying ATRA-resistant is genetic muta-
tions in the RARa ligand binding domain (75) which is not
related to the DDR, and impaired DDR are still observed in
ATRA-resistant cells (26). ZYH005 was identified accord-
ing to the results of specific cytotoxic activity to APL and
ATRA-resistant APL cells. Moreover, in ZYH005-treated
APL cells, ␥H2AX were increased sharply even at 1 h and
cleaved PARP were detectable at 3 h, but caspase-3 weakly
activated at 6 h, indicating that DNA damage is the earli-
est event in the cell after ZYH005 treatment. These findings
suggest that impairment of the DDR determined the re-
sponse of APL and ATRA-resistant APL cells to ZYH005
and reflecting a new strategy to overcome ATRA resistance
with DNA-binding drugs.
Several therapeutics have been tested to overcome ATRA
resistance, such as ATO, Am80 (76) and histone deacety-
lase inhibitors (77), among these, ATO is considered the
most active agent (78). The PML-RARa fusion protein
is clearly the central player driving APL, which also in-
duces strong resistance to apoptosis in APL cells (62).
ATO could directly target the PML component of PML-
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12 Nucleic Acids Research, 2018
RAR␣ and degrade it and then led to apoptosis of APL
cells. Similarly, ZYH005 treatment induces PML-RAR␣
degradation in APL and ATRA-resistant APL cells. The
difference is ATO degrade PML-RAR␣ mainly through
ubiquitin proteasome system (79,80), while ZYH005 in-
duces caspase-dependent PML-RAR␣ degradation. More-
over, ZYH005 induces apoptosis in both APL and ATRA-
resistant APL cells, and its apoptosis-inducing effect is more
potent than those of ATO and other widely used anti-
cancer drugs such as 5-fluorouracil, cisplatin and doxoru-
bicin. On the other hand, the caspase inhibitor Z-VAD-
FMK blocked ZYH005-induced PML-RAR␣ degradation
and apoptosis, but proteins related to the DNA damage
such as PARP and ␥H2AX, were partially blocked by Z-
VAD-FMK. Therefore, we conclude that ZYH005-induced
DNA damage activates caspase 3, the activated-caspase 3
cleaves the PML/RARa fusion protein subsequently facili-
tates APL and ATRA-resistant APL cells undergoing apop-
tosis. The precise mechanism of ZYH005-induced degra-
dation of PML-RAR␣ remains unclear; yet the apoptosis
inducing effect of ZYH005 offers a potent therapeutic ad-
vantage of ZYH005 and its role in overcoming ATRA resis-
tance warrants further validation in patient-derived samples
in future studies.
University, Professor Jie Yan of National University of Sin-
gapore for discussions and comments.
FUNDING
National natural Science Foundation of China [81503305
to Q.T. and 21708009 to H.Y.]; China Postdoctoral Science
Foundation funded project [2015M582227 to Q.T.]; Pro-
gram for Changjiang Scholars of Ministry of Education of
the People’s Republic of China [T2016088 to Y.Z.]; National
natural Science Foundation for Distinguished Young Schol-
ars [81725021 to Y.Z.]; Innovative Research Groups of the
National Natural Science Foundation of China [81721005
to Y.Z.]; Academic Frontier Youth Team of HUST; Inte-
grated Innovative Team for Major Human Diseases Pro-
gram of Tongji Medical College, HUST; Fundamental Re-
search Fund for the Central Universities [2017KFYXJJ153
to H.Y and 2017KFYXJJ152 to Z.L.]; Singapore Ministry
of Education Academic Research Fund Tier 3 [MOE 2012-
T3-1-001 to J.Y.]. Funding for open access charge: National
natural Science Foundation of China [81503305 to Q.T.].
Conflict of interest statement. None declared.
REFERENCES
Although ATO is used as the best salvage therapy agent
for ATRA-resistance patients, these patients subsequently
resistant to ATO have also been reported (23,81). Fur-
thermore, ATRA is a polyene compound whose sensitiv-
ity to light and heat may render it unstable, and the use
of ATO is associated with many acute adverse effects and
clinical complications, such as differentiation syndrome (7–
35%), pseudotumour cerebri, hyperleukocytosis, leukocyto-
sis (32–73%) and hepatic toxicity (74,82). Notably, ZYH005
possesses some advantages over the above agents, as the
drug has a simple, stable structure, inexpensive synthesis
procedure, and exerts negligible toxicity in vitro and in
vivo. Additionally, we observed that of the series of N-(4-
methoxylphenyl) ethyl derivatives, ZYH006 and ZYH007
showed activity similar to that of ZYH005 in the prelimi-
nary anticancer evaluation, indicating that the heteroatom
substituent in the core may determine the anticancer activ-
ity of these compounds; these findings as well as the po-
tential of intercalation in double-stranded DNA provide a
valuable resource for the field of biologically active phenan-
thridinone alkaloids.
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SUPPLEMENTARY DATA
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ACKNOWLEDGEMENTS
We are grateful to Professor Yingli Wu of Shanghai Jiao
Tong University for generously providing cells regarding
this study. We also thank Professor Hudan Liu of Wuhan
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