Synthesis of naphthazarin derivatives and identification of novel thioredoxin reductase inhibitor as potential anticancer agent

Article abstract of DOI:10.1016/j.ejmech.2017.09.027

Mammalian thioredoxin reductase (TrxR) enzymes play a crucial role in regulating multiple redox-based signaling pathways and have attracted increasing attention as promising anticancer drug targets. We report here the synthesis of a panel of naphthazarin derivatives and discovery of 2-methyl-5,8-dihydroxy-1,4-naphthoquinone (3, 2-methylnaphthazarin) as a potent cytotoxic agent with a submicromolar half maximal inhibitory concentration to the human promyelocytic leukemia HL-60 cells. Mechanism studies reveal that the compound selectively inhibits TrxR to induce oxidative stress-mediated apoptosis of HL-60 cells. Knockdown of TrxR sensitizes the cells to 3 insults, while overexpression of the functional enzyme confers resistance to the compound treatment, underpinning the physiological significance of targeting TrxR by 3. Clarification of the interaction of compound 3 with TrxR unveils a mechanism underlying the cellular action of the compound, and sheds light in considering development of the compound as a potential cancer chemotherapeutic agent.

Full text of DOI:10.1016/j.ejmech.2017.09.027

Accepted Manuscript
Synthesis of naphthazarin derivatives and identification of novel thioredoxin reductase
inhibitor as potential anticancer agent
Junmin Zhang, Yaping Liu, Danfeng Shi, Guodong Hu, Baoxin Zhang, Xinming Li,
Ruijuan Liu, Xiao Han, Xiaojun Yao, Jianguo Fang
PII:
S0223-5234(17)30741-9
10.1016/j.ejmech.2017.09.027
EJMECH 9742
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 April 2017
Revised Date: 15 September 2017
Accepted Date: 15 September 2017
Please cite this article as: J. Zhang, Y. Liu, D. Shi, G. Hu, B. Zhang, X. Li, R. Liu, X. Han, X. Yao,
J. Fang, Synthesis of naphthazarin derivatives and identification of novel thioredoxin reductase
inhibitor as potential anticancer agent, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.09.027.
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Graphical abstract

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Synthesis of naphthazarin derivatives and identification of novel thioredoxin
reductase inhibitor as potential anticancer agent
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Junmin Zhang a, b, c, Yaping Liu a, c , Danfeng Shi c, Guodong Hu a, c, Baoxin Zhang a, c, Xinming Li a, c
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Ruijuan Liu a, b, Xiao Han a, c, Xiaojun Yao c and Jianguo Fang a, c
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a State Key Laboratory of Applied Organic Chemistry, b School of Pharmacy, and c College of
Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000, China
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* Corresponding author. Tel: +86 931 8912500; fax: +86 931 8915557.
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E-mail: fangjg@lzu.edu.cn (J. Fang).
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Abstract
Mammalian thioredoxin reductase (TrxR) enzymes play a crucial role in regulating multiple
redox-based signaling pathways and have attracted increasing attention as promising anticancer drug
targets. We report here the synthesis of a panel of naphthazarin derivatives and discovery of
2-methyl-5,8-dihydroxy-1,4-naphthoquinone (3, 2-methylnaphthazarin) as a potent cytotoxic agent
with a submicromolar half maximal inhibitory concentration to the human promyelocytic leukemia
HL-60 cells. Mechanism studies reveal that the compound selectively inhibits TrxR to induce
oxidative stress-mediated apoptosis of HL-60 cells. Knockdown of TrxR sensitizes the cells to 3
insults, while overexpression of the functional enzyme confers resistance to the compound treatment,
underpinning the physiological significance of targeting TrxR by 3. Clarification of the interaction of
compound 3 with TrxR unveils the cellular action of the compound, and sheds light in considering
development of the compound as a potential cancer chemotherapeutic agent.
Keywords: Naphthazarin derivatives; 2-methylnaphthazarin; Thioredoxin reductase; Reactive
oxygen species; Oxidative stress; Apoptosis.
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Abbreviations: BSO: L-buthionine-(S,R)-sulfoximine; DCFH-DA: 2’, 7’-dichlorfluorescein diacetate; DHE:
dihydroethidium; GR: glutathione reductase; HeLa-shTrxR1: HeLa cells transfected with shTrxR1 plasmids;
HeLa-shNT: HeLa cells transfected with shNT plasmid; HEK-TrxR1: HEK 293T cells stably overexpressing TrxR1;
HEK-IRES: HEK 293T cells stably transfected with a vector; NAC: N-acetyl-L-cysteine; ROS: reactive oxygen
species; Sec: selenocysteine; Trx: thioredoxin; TrxR: thioredoxin reductase.
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1. Introduction
The thioredoxin system, consisting of NADPH, thioredoxin reductase (TrxR) and thioredoxin
(Trx), is a major disulfide reductase system that transfers electrons from NADPH to a multitude of
enzymes, and plays an essential role in regulating multiple cellular redox signaling pathways [1-3].
Elevated levels of Trx/TrxR are frequently observed in different types of cancer, and are recognized
as association with aggressive cancers, poor patient prognosis and resistance to chemotherapy [4, 5].
Furthermore, transfection with dominant-negative mutant Trx or depletion of TrxR results in a
retardation in tumor progression and metastasis [6, 7]. Accordingly, the thioredoxin system has been
a promising target for anticancer drug development [1, 8]. The past decades have witnessed growing
interests in identifying and developing small molecules targeting the TrxR/Trx as potential cancer
chemotherapeutic agents [8-16].
The diversity of natural products such as quinones, flavonoids, terpenoids, and alkaloids has
inspired the drug discovery, and is still a unique source of biologically active lead compounds [17,
18]. Approximately, half of the drugs in the market are natural products or their derivatives. In the
case of anti-infective and anticancer agents, this ratio is even higher [19]. As an essence of the drug
discovery progress, the generation of novel and structurally diverse lead compounds with potential
pharmacological properties has been actively sought. Among these natural products, 1, 4-
naphthoquinones are an important family. The 1,4-naphthoquinone pharmacophore is known to
afford antitumor activity in a number of clinically used drugs, such as streptonigrin [20], mitomycins
[21], and doxorubicin [22]. Naphthazarin (5, 8-dihydroxy-l, 4-naphthoquinone) is one of a natural 1,
4-naphthoquinone derivatives derived from the roots of several members of the genus Boraginaceae
[23] and is employed in food, cosmetics and textiles. Naphthazarin derivatives have been
documented possessing a wide variety of pharmacological activities due to containing a 1,
4-naphthoquinone pharmacophore, including antiinflammatory, anticancer, antibacterial, antioxidant,
and antifungal effects [24-27]. Ahn et al. synthesized a series of naphthazarin derivatives through
modifying 5, 8-dihydroxy, and evaluated their cytotoxicity and inhibition of DNA topoisomerase I
[28-30]. In recent years, large attention has been paid to the potential anticancer potency of
naphthazarin derivatives due to induction of apoptosis in multiple types of tumor cells, and various
putative cellular mechanisms were proposed, including oxidative stress [24], depolymerization of
microtubules [25], interference with lysosomal function [31], activation of mitochondrial apoptosis
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inducing factor (AIF) [32] and p53-dependent p21 activation [33]. Shikonin, a naturally occurring
naphthazarin derivative, was previously investigated for killing cancer cells via targeting TrxR by
our lab [15]. In consideration of the importance of 5, 8-dihydroxy-l, 4-naphthoquinone scaffold, we
synthesized a series of 2-substituted naphthazarin derivatives, and evaluated their cytotoxicity in a
panel of tumor cells and normal cells. After initial cytotoxicity screening, we discovered that 3
(2-methylnaphthazarin) displayed the most cytotoxic effect against HL-60 cells with an IC50 value of
0.72 µM after 48 h treatment. The follow-up studies disclosed that 3 results in a time- and
concentration-dependent inhibition of the TrxR. The pharmacological action of 3 in cells involves
inhibition of TrxR, accumulation of reactive oxygen species (ROS), decrease of GSH/GSSG ration
and cellular total thiols, and activation of caspase 3. Finally, compound 3 elicits oxidative
stress-mediated apoptosis of HL-60 cells. Knockdown of TrxR sensitizes the cells to 3 insults, while
overexpression of the functional enzyme alleviates the cytotoxicity, underpinning the physiological
significance of targeting TrxR by 3. We expect that 3 could be a novel cancer chemotherapeutic
agent for further development.
2. Results
2.1. Chemistry
Synthesis of the naphthoquinone derivatives was performed by condensation of maleic
anhydrides with various phenols under a molten mixture of aluminium chloride and sodium chloride
[34]. The general synthetic routes were depicted in Scheme 1. After construction of the naphthazarin
(2) scaffold, various substituted naphthazarin were prepared via reactions of 2 with different amines
in ethanol as described in the literatures [35, 36]. The resulting products were purified by silica gel
column chromatography. All the naphthoquinone derivatives were fully characterized by 1H, 13C
NMR, and mass spectrometry. The original NMR spectra of compounds were shown in the
Supporting Information (Fig. S1-S30).
2.2. Evaluation of cytotoxicity.
Initially, we employed the MTT assay to assess the cytotoxicity of all the synthetic compounds
(1-16, structures presented in Scheme 1 and Table 1) towards HL-60 cells at a fixed concentration of
20 µM over a duration of 48 h. Under this condition, the cell viability was summarized and listed in
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Table 1. Compounds 2 and 3 showed the highest cytotoxicity. Analyzing the cytotoxicity data reveals
that introducing a substituted amino group to the quinone double bond (compounds 4-16)
significantly decreases the cytotoxicity, which provides information for further preparation of such
compounds. Next, we chose these two compounds for the follow-up studies. As shown in Fig.1A,
compounds 2 and 3 display comparable efficacy toward HL-60 tumor cells with a submicromolar
IC50 value after 48 or 72 h treatment, but compound 3 appears higher potency. As compounds 2 and 3
have similar structures, it is likely that they share the same action mechanism in cells. Thus, we
chose 3 to confirm the results from the MTT assay by employing the trypan blue exclusion assay,
which gives an IC50 value of 0.86 µM (Fig. 1C) upon treatment of HL-60 cells for 48 h. This is quite
consistent with the value from the MTT assay (IC50=0.72 µM). To confirm whether 3 is cytotoxic to
other tumor cells and normal cells, several human cancer and noncancerous cell lines were treated
with various concentrations of 3 for 48 h, including human alveolar adenocarcinoma cell line A549,
human cervical cancer cell line HeLa, human hepatocellular carcinoma cell line HepG2, human
embryonic kidney 293T cell line HEK 293T and human immortalized normal liver cell line L02. As
shown in Fig. 1B, compound 3 exhibited the most potency on HL-60 cells compared with other cell
types. Interestingly, compound 3 displays less potency to L02 cells and HEK 293T cells, two
noncancerous cell lines (Fig. 1D). Collectively, our results demonstrate that compound 3 possesses
the promising cytotoxicity in all compounds and inhibits growth of multiple cell lines, with the most
potency to HL-60 cells. Thus, our follow-up studies were focused on 3.
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2.3. Inhibition of TrxR by 3 in vitro
Since 3 displays the highest cytotoxicity to HL-60 cells, we then asked the possible cellular
target of the compound. Compound 3 belongs to the naphthoquinone family, a class of reactive
natural products whose chemical structures allow them to interact with pharmacological targets by
forming covalent bonds and/or by serving on electron transfer agents in biological redox reactions
[37]. More importantly, its structure is reminiscent of shikonin [15], and juglone [38], two
established TrxR inhibitors. Thus we speculated that 3 might be a novel inhibitor of TrxR. Initially,
we determined whether compound 3 inhibits TrxR using the purified enzyme. Incubation of 3 with
the reduced enzyme causes a dose-dependent inhibition of its activity with an IC50 value around 0.7
µM (Fig. 2A), which is more potent than other known TrxR inhibitors, such as securinine [14],
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shikonin [15], and parthenolide [16] . Removal of 3 from the incubation mixture by desalting column
did not recover the enzyme activity, suggesting compound 3 inhibits TrxR1 irreversibly. TrxR
possess an N-terminal redox center characterized by a dithiol motif (-Cys-XXXX-Cys-) and a
C-terminal active site with a -Cys-Sec- motif. The Sec residue has a significantly low pKa value
(5.24 for the selenol/selenate couple compared to 8.25 for the thiol/thiolate couple), resulting in an
enhanced nucleophilic property [39, 40]. As many TrxR inhibitors have been demonstrated to target
the Sec residue in the enzyme [11, 13, 14, 41], next, we determined whether 3 also targets the Sec
residue by comparing the inhibition potency of 3 towards the wild type enzyme and the mutant
U498C TrxR enzyme, where the Sec498 was replaced by Cys. As shown in Fig. 2A, compound 3
gave very weak inhibition to U498C TrxR. Glutathione reductase (GR) is a homologous of TrxR
with a similar overall structure but lacks the C-terminal -Cys-Sec- redox center. Compound 3 had
marginal effect on the enzyme activity (Fig. 2A). Selectively inhibiting WT enzyme but not U498C
TrxR or GR implies the Sec residue is specifically targeted by 3. In addition, the possible binding
mode for compound 3 with TrxR protein was figured out by the covalent docking method. As shown
in Fig. 2B, the carbon atom indicated with an asterisk of compound 3 connects covalently with the
selenium atom of Sec498. The binding pocket for compound 3 was formed by the C-terminal active
site redox center of one subunit constituted by Gly496, Cys497, Sec498, Gly499 and some
hydrophobic residues of the other subunit including Val60’, Ile65’, Leu112’, Ile347’. A hydrogen
bonding was found between the phenolic hydroxyl group of compound 3 and the carboxyl group of
Gly499 and a π-π stacking interaction also occurs between the benzene ring of compound 3 and the
imidazolyl group of His472. All these results presented a clear view that compound 3 reacts with
Sec498 of TrxR irreversibly by forming a covalent bond and all the non-bonding interactions
between compound 3 and the residues around Sec498 seems to supply a favorable environment for
the formation of the covalent bond. Taken together, compound 3 selectively targets the Sec residue
in TrxR to inhibit its activity in vitro.
2.4. Inhibition of TrxR contributes to the cytotoxicity of 3
Since compound 3 effectively inhibits the purified TrxR, to extend this discovery in a cellular
content, we determined the inhibition of TrxR in HL-60 cells. Firstly, we employed the TRFS-green,
a specific TrxR fluorescence probe developed by our lab [42], to assess the cellular TrxR activity. As
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illustrated in Fig. 3A, treatment of HL-60 cells with 3 causes a dose-dependent decrease of the
fluorescence, demonstrating the inhibition of TrxR by 3. Secondly, we performed the classic
Trx-mediated insulin reduction assay to confirm the fluorescence results. Again, treatment of HL-60
cells with 3 causes a remarkable decrease in the cellular TrxR activity with an IC50 value around 2.4
µM (Fig. 3B), which is more potent than other known TrxR inhibitors, such as securinine [14],
parthenolide [16], and alantolactone [41]. There appears no apparent change of TrxR1 expression
after the HL-60 cells were treated with 3 for 24 h (Fig. 3C), suggesting that the decreased enzyme
activity is caused by the direct inhibition of TrxR. Finally, to further demonstrate whether the
inhibition of TrxR is involved in the cytotoxicity of 3, we then turned to compare the sensitivity of
the cells that stably transfected with a vector (HEK-IRES) and HEK cells stably overexpressing
TrxR1 (HEK-TrxR1) towards 3 treatment. The transfection efficiency was confirmed by measuring
the enzyme activity and the protein expression in our previous publication [41]. As shown in Fig. 3D,
3 shows lower cytotoxicity to HEK-TrxR1 cells than HEK-IRES cells. Meanwhile, to further verify
the biological relevance of TrxR-mediated cytotoxicity of 3, we further determined the cytotoxicity
of 3 after knocking down TrxR expression. Due to the low transfection efficiency of HL-60 cells, we
employed the HeLa cells for the knockdown experiments. Knockdown of TrxR1 in HeLa cells
(HeLa-shTrxR1) was assessed in our group previous publication [41]. TrxR knockdown could yield
drug-specific alteration of cytotoxicity of small molecule drugs [43]. We observed that 3 displays
elevated potency to HeLa-shTrxR1 cells compared with the cells only transfected with a
non-targeting vector (HeLa-shNT) (Fig. 3E). Taken together, our results revealed that inhibition of
TrxR is involved in the cellular action of 3.
2.5. Induction of oxidative stress by 3 in HL-60 cells
The thioredoxin system is critical for the intracellular redox balance to prevent excess ROS
accumulation. A great majority of TrxR inhibitors, such as curcumin [44] and shikonin [15], could
promote ROS production and induce oxidative stress. Accordingly, we determined whether
compound 3 has the similar effects. Firstly, DCFH-DA was employed to assess the overall ROS
levels in HL-60 cells. DCFH-DA is a well-established probe to determine intracellular levels of ROS.
As shown in Fig. 4A, the ROS levels in HL-60 cells are undetectable under basal conditions.
Treatment of the HL-60 cells with 3 notably promotes the ROS production. The higher concentration
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of 3 leads to more ROS generation. The DCFH-DA-based ROS assay is prone to artifacts as
extensively discussed in the literature [45], we further employed another probe, DHE, to confirm the
ROS generation in HL-60 cells. Again, compound 3 evokes the fluorescence in a dose-dependent
manner (Fig. 4B). Collectively, these data indicated that 3 could promote ROS accumulation in
HL-60 cells.
Cellular thiols are important antioxidants to neutralize ROS accumulation. To assess the overall
redox states of the HL-60 cells after 3-treatment, the amount of thiols was determined by DTNB
titration. After HL-60 cells were treated with 3, the amount of cellular thiols is decreased (Fig. 4C).
Furthermore, we assessed the changes of glutathione homeostasis in HL-60 cells treated with the
indicated concentration of 3. As shown in Fig. 4D, incubation of 3 with the cells causes a reduction
of the ratio of GSH/GSSG dose-dependently. Accumulation of ROS and oxidation of thiols indicate
the intracellular environment is shifted to an oxidative state. Thus, we further asked if the additional
oxidative stimulants could improve the cytotoxicity of 3. As shown in Fig. 4E, pretreatment of the
HL-60 cells with 3 significantly incited the cells to hydrogen peroxide challenge. Under our
experimental conditions, hydrogen peroxide (50 µM) and 3 (1 µM) caused 4% and 27% cell death,
respectively. However, treatment with a combination of hydrogen peroxide and 3 stimulates more
than 43% cell death, indicating that 3 has synergistic action with hydrogen peroxide to induce cell
death. A similar effect was observed for the combination of 3 µM of 3 with hydrogen peroxide.
Taken together, all these data support the induction of oxidative stress by 3 in HL-60 cells.
2.6. Promotion of cell death by GSH depletion and protection of cell death by NAC
As 3 could lead to disorder of cellular redox balance and induction of oxidative stress in HL-60
cells, we next explored the functions of GSH depletion and NAC supplement in the process of
3-induced HL-60 cell death. NAC is a thiol antioxidant and precursor for biosynthesis of GSH.
Pretreatment of the HL-60 cells with NAC alleviates the cytotoxicity of 3, and a high concentration
of NAC (5 mM) completely rescues the cells (Fig. 5A). Next, we measured the effect of GSH
depletion on the cytotoxicity of 3. In contrast to the protective role of NAC, depletion of the cellular
GSH by pretreatment of the cells with BSO remarkably augments the cytotoxicity of 3 (Fig. 5B).
Under our experimental conditions, the intracellular GSH level was reduced to less than 20% of the
control after treatment of HL-60 cells with 50 µM BSO for 24 h. GSH is a crucial component of
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glutathione system, another redox regulation network besides thioredoxin system in cells. Our results
that depletion of cellular GSH sensitizes the cytotoxicity while supplement of NAC protects the cells
to 3 treatment support the oxidative stress is engaged in the pharmacological action of 3.
2.7. Induction of apoptosis in HL-60 cells
We presented evidence here that 3 kills HL-60 cells predominantly through the induction of
apoptosis (Fig. 6). Since the apoptotic process is an early event of cell death, we chose a shorter
treatment time (24 h) for the apoptosis assay. It is well known that caspase 3 is a major component of
apoptotic machinery in mammalian cells, and its activation is one of hallmarks in the process of
apoptosis. We detected the activation of caspase 3 after 3 treatment. As shown in Fig. 6A, compound
3 activates caspase 3 activity in HL-60 cells. However, a higher concentration of 3 (5 µM) decreased
the activity of caspase 3 slightly. This could be due to inactivation of caspase 3 by the high
concentration of 3. When HL-60 cells were incubated with indicated concentrations of 3 followed by
Hoechst 33342 staining, an increasing number of cells exhibited condensed and highly fluorescent
nuclei, a characteristic morphology of cells undergoing apoptosis (Fig. 6B). Further evidence from
the Annexin V-FITC/PI double staining assay quantified the apoptotic population by flow cytometer
(Fig. 6 C&D). Treatment of the cells with 0.5 µM, 1 µM, 3 µM and 5 µM 3 for 24 h causes ~10%,
~23%, ~42% and ~56% cell apoptosis, respectively. Again, NAC could antagonize the apoptosis. A
slight increase of the necrotic cells was observed after the cells were treated with 5 µM 3 for 24 h,
consistent with the higher concentration of 3 (5 µM) decrease the activation of caspase 3 (Fig. 6A).
This could be due to the excessive oxidative stress caused by 3. Taken together, our results reveal
that compound 3 predominantly induces apoptosis in HL-60 cells.
3. Discussion
l, 4-Naphthoquinone moiety is believed to be an essential pharmacophore of many
pharmaceutically active compounds. We have synthesized a series of 2-substituted l, 4-
naphthoquinone derivatives, evaluated their anticancer activity, and explored the underlying
mechanism. Among these compounds, compound 3 inhibits the growth of multiple cell lines, with
the most potency to HL-60 cells. We presented evidence herein that the cytotoxicity of 3 is mediated,
at least in part, by targeting the selenoenzyme TrxR. Firstly, we compared the inhibition potency of 3
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to the purified WT TrxR1, GR and U498C TrxR1 (Fig. 2A). Single mutation of Sec to Cys sharply
decreased the potency of 3, suggesting that the Sec residue in TrxR is a primary target of 3. The
structure of GR is closely related to TrxR, however, under our experimental conditions, very weak
inhibition of GR was observed. The selective inhibition of WT TrxR1 but not U498C TrxR1 nor GR
suggests a specific interaction of 3 with TrxR. Secondly, we also investigated the binding mode of
compound 3 with TrxR using the covalent docking method (Fig. 2B), supporting the covalent bond
formation between Sec498 and compound 3. Binding of 3 to the enzyme is further supported by a
series of non-bonding interactions between the compound and the residues around Sec498. Thirdly,
we provided evidence in cellular contexts to support the unique role of TrxR for the cellular action of
3. Compound 3 displays remarkable inhibition of TrxR activity in intact cells and in cell lysates (Fig.
3A & B). More importantly, involvement of TrxR inhibition to the cytotoxicity by 3 was further
demonstrated by the TrxR overexpression and knockdown experiments (Fig. 3D & E). Under our
experimental conditions, compound 3 displays potent cytotoxicity to a variety of cancer cells, but has
the highest potency to HL-60 cells (Fig. 1B). We reasoned this selectivity may be related to the
intrinsic TrxR activity in different cells. HL-60 cells, which are most sensitive to 3 among the four
tested cell lines, show the lowest TrxR activity [15]. TrxR knockdown experiments further confirm
this result (Fig. 3 E). Finally, the glutathione system and thioredoxin system are two major networks.
Although they work independently, a few crosstalks enable them acting as mutual backup in
managing the intracellular redox homeostasis [46]. Our observations that depletion of cellular GSH
by BSO elevates the cytotoxicity, while upregulation of GSH by NAC lessens the cytotoxicity of 3
(Fig. 5A & B) also suggest that TrxR is involved in the cellular action of 3. Inhibition of TrxR would
further promote accumulation of ROS, decrease cellular thiol pool, and finally elicit oxidative stress.
Acquiring resistance toward apoptosis is one of the hallmarks of malignant cells [47].
Activation of apoptosis in tumor cells is thus indispensable to tumor therapy. We provided evidence
that the cell death caused by 3 is predominantly through apoptosis (Fig. 6), thereby strengthening the
application of 3 in treatment of cancer. In addition, as we have demonstrated that treatment of the
cells with 3 promotes accumulation of ROS, decrease of GSH/GSSG ration and depletion of
intracellular total thiols in HL-60 cells, it is most likely that 3 induces oxidative stress-mediated
apoptosis. Based on these results shown, we here presented that targeting TrxR by 3 contributes to
inducing oxidative stress-mediated apoptosis of HL-60 cells.
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In summary, we have synthesized a series of l, 4-naphthoquinone derivatives and discovered
compound 3 as a potent cytotoxic agent with a submicromolar half maximal inhibitory concentration
to HL-60 cells. The follow-up mechanistic studies disclosed that the cellular action of compound 3 is
related to its ability to target the selenoenzyme TrxR. Further investigation revealed that 3 could
induce ROS production and thiol depletion, and promote oxidative stress-mediated apoptosis in
HL-60 cells. Clarification of the interaction of compound 3 with TrxR unveils the cellular target of
the compound, and sheds light in considering development of it as a potential cancer
chemotherapeutic agent.
4. Experimental section
4.1. Materials
The recombinant rat TrxR1 (WT TrxR1) was a gift from Prof. Arne Holmgren (Karolinska
Institute, Sweden). The E. coli Trx and recombinant U498C TrxR1 mutant (Sec→Cys) were
produced as described [12]. The shRNA plasmids targeting TrxR1 (shTrxR1) and non-targeting
control (shNT), and HEK 293T cells stably transfected with a vector (HEK-IRES) and the cells
stably overexpressing TrxR1 (HEK-TrxR1) were gifts from Prof. Constantinos Koumenis
(University of Pennsylvania School of Medicine). All pictures of cell imaging were acquired by a
FLoid Cell Imaging Station (Thermo Fisher Scientific). N-acetyl-L-cysteine (NAC), L-buthionine-(S,
R)-sulfoximine (BSO), bovine insulin, N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA),
G418, 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), reduced
glutathione (GSH), glutathione disulfide (GSSG), yeast glutathione reductase (GR), puromycin,
dimethyl sulfoxide (DMSO), 2’, 7’-dichlorfluorescein diacetate (DCFH-DA), Dulbecco's modified
Eagle’s medium (DMEM), RPMI 1640 medium and Hoechst 33342 were obtained from
Sigma-Aldrich (St. Louis, USA). NADPH was purchased from Roche (Mannheim, Germany). Fetal
bovine serum (FBS) was obtained from Sijiqing (Hangzhou, China). The PVDF membrane was from
Millipore (Billerica, MA). Penicillin, streptomycin, and 3-(4, 5-Dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT) were obtained from Amresco (Solon, OH). Bovine serum
albumin (BSA), sodium orthovanadate (V) (Na3VO4), trypan blue phenylmethylsulfonyl fluoride
(PMSF), and actin antibody (AA128-1) were obtained from Beyotime (Nantong, China). The
apoptosis detection kit containing propidium iodide (PI) and fluorescein-5-isothiocyanate-conjugated
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Annexin V (Annexin V-FITC) was from Zoman Biotech (Beijing, China). DTNB was obtained from
J&K Scientific (Beijing, China). TrxR1 primary antibody (sc-28321), anti-mouse IgG-HRP (sc-2031),
anti-rabbit IgG-HRP (sc-2004) and dihydroethidium (DHE) were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). 1H NMR and 13C NMR spectra were recorded with a Bruker AMX
spectrometer at 400 and 100 MHz, respectively, using TMS as the internal standard. Mass spectra
were recorded on Trace DSQ GC-MS spectrometer. All other reagents were of analytical grade and
were purchased from commercial supplies. All reactions were carried out under a deoxygenated and
dry argon atmosphere unless otherwise indicated.
4.2. General method for the preparation of naphthazarins
To a mixture of anhydrous AlCl3 (10 mmol, 1.33 g) and NaCl (5 mmol, 0.29 g) that was heated
in an oil bath till molten was added maleic anhydride or 2-methylmaleic anhydride (1 mmol) and 1,
4-benzenediol or 4-chlorophenol (1 mmol). The temperature was slowly increased and maintained at
165 oC for 4 h. The reaction mixture was cooled to 0 oC, and 10 mL of 10% HCl was added, and the
mixture was stirred for 15 min at 0 °C and then refluxed for 30 min. The reaction mixture was cooled
to room temperature and extracted with ethyl acetate. The resulting product was purified by silica gel
column chromatography using petroleum ether and ethyl acetate as the mobile phase. For preparation
of substituted naphthazarins, compound 2 (114 mg, 0.6 mmol) was stirred in EtOH (10 mL), and
different amines (0.7 mmol) were added. The reaction was allowed to stir for several hours at room
temperature and the reaction process was monitored by thin layer chromatography. After the reaction
completed, the solvent was evaporated under vacuum, and the resulting product was purified by
silica gel column chromatography using petroleum ether and ethyl acetate as the mobile phase.
4.2.1. 5-chloro-8-hydroxy-1, 4-naphthoquinone (1)
Yield: 22%. 1H NMR (400 MHz, CDCl3): δ 12.62 (s, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.25 (d, J =
8.8 Hz, 1H), 6.95 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 189.8, 182.7, 161.3, 140.9, 140.4, 136.9,
126.8, 126.4, 125.5, 116.0. EI-MS m/z (%): 208 (M+, 100), 207 (32), 210(31), 152 (23), 57 (21), 71
(21), 126 (20), 85 (19), 44 (16).
4.2.2. 5, 8-dihydroxy-1, 4-naphthoquinone (2)
Yield: 15%. 1H NMR (400 MHz, DMSO): δ 12.22 (s, 2H), 7.28 (s, 4H). 13C NMR (100 MHz,
DMSO): δ 172.2, 134.5. EI-MS m/z (%): 190 (M+, 100), 189 (49).
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4.2.3. 2-methyl-5, 8-dihydroxy-1, 4-naphthoquinone (3)
Yield: 20%. 1H NMR (400 MHz, CDCl3): δ 12.57 (s, 1H), 12.45 (s, 1H), 7.21 (m, 2H), 6.90 (s,
1H), 2.24 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 183.3, 182.9, 162.9, 162.3, 148.2, 135.2, 131.2,
130.9, 111.8, 16.2. EI-MS m/z (%): 204 (M+, 100), 189 (22).
4.2.4. 2-methylamino-5, 8-dihydroxy-1, 4-naphthoquinone (4)
Yield: 85%. 1H NMR (400 MHz, DMSO): δ13.70 (s, 1H), 11.68 (s, 1H),8.09 (m, 1H), 7.32 (d, J
= 9.2 Hz, 1H), 7.20 (d, J = 9.2 Hz, 1H), 5.60 (s, 1H), 2.84 (s, 3H).13C NMR (100 MHz, DMSO): δ
186.1, 183.6, 156.3, 155.0, 150.6, 130.7, 125.7, 111.7, 111.1, 98.6, 29.2. EI-MS m/z (%): 219 (M+,
100), 218 (23), 149(18).
4.2.5. 2-ethylamino-5, 8-dihydroxy-1, 4-naphthoquinone (5)
Yield: 89%. 1H NMR (400 MHz, DMSO): δ13.71 (s, 1H), 11.69 (s, 1H), 8.01 (m, 1H), 7.32 (d,
J = 9.2 Hz, 1H), 7.20 (d, J = 9.2 Hz, 1H), 5.68 (s, 1H), 3.28 (q, 2H), 1.18 (t, 3H).13C NMR (100
MHz, DMSO): δ 186.1, 183.8, 156.3, 154.9, 149.4, 130.7, 125.6, 111.7, 111.0, 98.5, 36.9, 12.9.
EI-MS m/z (%): 233 (M+, 95), 218 (100).
4.2.6. 2- propylamino-5, 8-dihydroxy-1, 4-naphthoquinone (6)
Yield: 88%. 1H NMR (400 MHz, DMSO): δ13.72 (s, 1H), 11.70 (s, 1H), 8.05 (m, 1H), 7.33 (d,
J = 9.2 Hz, 1H), 7.20 (d, J = 9.2 Hz, 1H), 5.70 (s, 1H), 3.22 (t, 2H), 1.64 (m, 2H), 0.92 (t, 3H). 13C
NMR (100 MHz, DMSO): δ 186.1, 183.7, 156.3, 154.9, 149.7, 130.7, 125.6, 111.7, 111.0, 98.5, 43.7,
20.7, 11.3. EI-MS m/z (%): 247 (M+, 47), 218 (100), 149 (25), 219 (21).
4.2.7. 2-n-butylamino-5, 8-dihydroxy-1, 4-naphthoquinone (7)
Yield: 85%. 1H NMR (400 MHz, DMSO): δ13.71 (s, 1H), 11.70 (s, 1H), 8.03 (m, 1H), 7.32 (d,
J = 9.2 Hz, 1H), 7.19 (d, J = 9.2 Hz, 1H), 5.67 (s, 1H), 3.24 (t, 2H), 1.58 (m, 2H), 1.36 (m, 2H), 0.92
(t, 3H).13C NMR (100 MHz, DMSO): δ 186.1, 183.7, 156.3, 154.9, 149.6, 130.7, 125.6, 111.7, 111.0,
98.5, 41.8, 29.4, 19.6, 13.6. EI-MS m/z (%): 261 (M+, 33), 44 (100), 218 (81), 57 (35), 149 (29), 194
(24).
4.2.8. 2- n-hexylamino-5, 8-dihydroxy-1, 4-naphthoquinone (8)
Yield: 87%. 1H NMR (400 MHz, DMSO): δ13.71 (s, 1H), 11.69 (s, 1H), 8.03 (m, 1H), 7.32 (d,
J = 9.2 Hz, 1H), 7.19 (d, J = 9.2 Hz, 1H), 5.67 (s, 1H), 3.21 (t, 2H), 1.58 (m, 12H), 1.28 (m, 10H),
0.86 (t, 3H). 13C NMR (100 MHz, DMSO): δ 186.1, 183.7, 156.3, 154.9, 149.6, 130.7, 125.6, 111.7,
111.0, 98.5, 42.1, 31.2, 28.7, 28.6, 27.2, 26.4, 22.0, 13.9. EI-MS m/z (%): 317 (M+, 33), 218 (100),
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156 (24), 219 (23).
4.2.9. 2-hydroxyethylamino-5, 8-dihydroxy-1, 4-naphthoquinone (9)
Yield: 89%. 1H NMR (400 MHz, DMSO): δ13.68 (s, 1H), 11.68 (s, 1H), 7.79 (m, 1H), 7.33 (d,
J = 9.2 Hz, 1H), 7.21 (d, J = 9.2 Hz, 1H), 5.75 (s, 1H), 4.90 (m, 1H), 3.62 (m, 2H), 3.31 (m, 2H).
13C NMR (100 MHz, DMSO): δ 186.3, 183.6, 156.3, 154.9, 149.9, 130.8, 125.7, 111.6, 110.9, 98.9,
58.4, 44.8. EI-MS m/z (%): 249 (M+, 35), 218 (100), 219 (15).
4.2.10. 2-isobutylamino -5, 8-dihydroxy-1, 4-naphthoquinone (10)
Yield: 85%. 1H NMR (400 MHz, DMSO): δ13.72 (s, 1H), 11.71 (s, 1H), 7.62 (m, 1H), 7.33 (d,
J = 9.2 Hz, 1H), 7.20 (d, J = 9.2 Hz, 1H), 5.72 (s, 1H), 3.58 (m, 1H), 1.70 (m, 1H), 1.57 (m, 1H),
1.19 (d, J = 6.4 Hz, 2H), 0.89 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO): δ 186.1, 183.7,
156.2, 154.8, 148.9, 130.6, 125.5, 111.6, 110.8, 98.5, 49.5, 27.8, 18.7, 10.4. EI-MS m/z (%): 261 (M+,
47), 232 (100), 71 (24), 57 (23), 85 (21).
4.2.11. 2-isobutylamino-5, 8-dihydroxy-1, 4-naphthoquinone (11)
Yield: 80%. 1H NMR (400 MHz, DMSO): δ13.70 (s, 1H), 11.69 (s, 1H), 8.00 (m, 1H), 7.32 (d,
J = 9.2 Hz, 1H), 7.19 (d, J = 9.2 Hz, 1H), 5.65 (s, 1H),3.25 (m, 2H), 1.67 (m, 1H),1.48 (m, 2H), 0.92
(d, J = 6.8 Hz, 6H). 13C NMR (100 MHz, DMSO): δ 186.0, 183.7, 156.3, 154.9, 149.5, 130.7, 125.6,
111.7, 111.0, 98.5, 40.4, 35.9, 25.4, 22.3. EI-MS m/z (%): 275 (M+, 36), 218 (100), 219 (54), 178
(19).
4.2.12. 2-phenylamino-5, 8-dihydroxy-1, 4-naphthoquinone (12)
Yield: 82%. 1H NMR (400 MHz, DMSO): δ13.36 (s, 1H), 11.78 (s, 1H), 9.59 (s, 1H), 7.49 (m,
2H), 7.40 (m, 3H), 7.29 (m, 2H), 6.55 (s, 1H), 6.01 (s, 1H). 13C NMR (100 MHz, DMSO): δ 187.2,
183.7, 156.4, 154.9, 149.7, 147.6, 137.6, 130.7, 129.4, 126.4, 125.9, 124.1, 115.6, 111.7, 110.9, 101.3.
EI-MS m/z (%): 281 (M+, 40), 149 (100), 45 (83).
4.2.13. 2- phenylmethylamino-5, 8-dihydroxy-1, 4-naphthoquinone (13)
Yield: 81%. 1H NMR (400 MHz, DMSO): δ13.54 (s, 1H), 11.71 (s, 1H), 8.60 (m, 1H), 7.35 (m,
3H), 7.32 (d, J = 9.3 Hz, 1H), 7.28 (m, 1H), 7.21 (d, J = 9.3 Hz, 1H), 5.59 (s, 1H), 4.50 (d, J = 6.4
Hz, 1H). 13C NMR (100 MHz, DMSO): δ 186.3, 183.7, 156.3, 155.0, 149.6, 137.0, 130.7, 128.5,
127.2, 127.1, 125.9, 111.7, 110.9, 99.8, 45.2. EI-MS m/z (%): 295 (M+, 67), 91 (100), 129 (50), 204
(34).
4.2.14. 2-piperidyl-5, 8-dihydroxy-1, 4-naphthoquinone (14)
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Yield: 89%. 1H NMR (400 MHz, DMSO): δ13.35 (s, 1H), 11.70 (s, 1H), 7.30 (d, J = 9.2 Hz,
1H), 7.21 (d, J = 9.2 Hz, 1H), 6.00 (s, 1H), 3.57 (m, 4H), 1.65 (m, 6H). 13C NMR (100 MHz,
DMSO): δ 186.1, 185.3, 155.9, 154.5, 154.1, 129.2, 126.4, 113.4, 111.1, 108.4, 50.3, 25.5, 23.6.
EI-MS m/z (%): 273 (M+, 100), 149 (95), 43 (70), 57 (69), 83 (66).
4.2.15. 2-morpholin-5, 8-dihydroxy-1, 4-naphthoquinone (15)
Yield: 88%. 1H NMR (400 MHz, DMSO): δ13.22 (s, 1H), 11.73 (s, 1H), 7.32 (d, J = 9.2 Hz,
1H), 7.24 (d, J = 9.2 Hz, 1H), 6.06 (s, 1H), 3.74 (m, 4H), 3.61 (m, 4H). 13C NMR (100 MHz,
DMSO): δ 186.7, 185.0, 156.1, 154.7, 154.0, 129.4, 126.8, 113.2, 111.0, 109.6, 65.7, 49.4. EI-MS
m/z (%): 275 (M+, 10), 149 (100), 167 (35), 57 (30).
4.2.16. 2-(pyrrolidin-1-yl)-5, 8-dihydroxy-1, 4-naphthoquinone (16)
Yield: 80%. 1H NMR (400 MHz, DMSO): δ13.79 (s, 1H), 11.86 (s, 1H), 7.30 (d, J = 9.2 Hz,
1H), 7.19 (d, J = 9.2 Hz, 1H), 5.64 (s, 1H), 3.98 (m, 2H), 3.38 (m, 2H), 1.94 (m, 4H). 13C NMR (100
MHz, DMSO): δ 185.5, 185.0, 156.1, 154.4, 149.5, 129.7, 125.6, 112.6, 111.1, 103.0. EI-MS m/z (%):
259 (M+, 100), 190 (24), 149 (19).
4.3. Cell lines and culture conditions
HL-60, HepG2, HeLa, HEK 293T and L02 cells were obtained from the Shanghai Institute of
Biochemistry and Cell Biology, Chinese Academy of Sciences. HepG2, HeLa, HEK 293T and L02
cells were kept in DMEM with 10% FBS, 2 mM glutamine, and 100 units/ml penicillin/streptomycin
and maintained in an atmosphere of 5% CO2 at 37 °C. HL-60 cells were kept in RPMI 1640
supplemented 10% FBS under the same conditions. HeLa-shNT and HeLa-shTrxR1 cells were
generated in our lab [38] and were cultured under the same conditions as those of HeLa cells with
additional supplement of puromycin (1 µg/ml) in DMEM. HEK-IRES and HEK-TrxR1 cells were
cultured under the same conditions as those of HEK 293T cells with additional supplement of 0.1
µM sodium selenite and 0.4 mg/ml G418 in DMEM.
4.4. Cell viability analysis.
4.4.1. MTT assay
Cells (1×104 ) were incubated with compounds in triplicate in a 96-well plate for indicated times
at 37 °C in a final volume of 100 µL. Controls were treated with 0.1% DMSO alone. At the end of
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the treatment, 10 µl of MTT (5 mg/ml) was added and continued incubating for additional 4 h. After
addition of the extraction buffer (100 µl, 10% SDS, 5% iso-butanol, 0.1% HCl), cells were further
incubated overnight. The absorbance was determined at 570 nm using a microplate reader (Thermo
Scientific Multiskan GO, Finland). Data are calculated as cell viability (% control) and corresponds
to the percentage viable cells compared to untreated cells.
4.4.2. Trypan blue exclusion assay
HL-60 cells (5×104 ) were incubated with 3 or other agents in 24-well plates for indicated times
at 37 °C. Controls were treated with DMSO alone, and cell viability was measured by the trypan blue
exclusion assay. After treatment indicated times, the cells were stained with trypan blue (0.4%, w/v),
and the number of dead (stained) and viable (non-stained) cells were determined using a microscope.
4.5. Purified TrxR activity assay
The purified TrxR activity was measured by the DTNB reduction assay. NADPH-reduced TrxR
(85 nM) or U498C TrxR (350 nM) was incubated with varying concentrations of 3 for 1 h at room
temperature in a 96-well plate (50 µL). A master mixture containing NADPH and DTNB in TE
buffer (50 mM Tris-HCl, 1 mM EDTA, pH 7.5, 50 µL) was added. The final concentrations of
DTNB and NADPH in the mixture are 2 mM and 200 µM, respectively. The linear increase of
optical density at 412 nm from the initial 3 min was read. TrxR activity was expressed as percentage
of the control.
4.6. GR assay
The NADPH-reduced GR (0.25 units/ml) in TE buffer was incubated with varying
concentrations of 3 for 1 h in a 96-well plate at room temperature in a total volume of 100 µL.
Reactions were initiated by the addition of NADPH (final concentration 400 µM) and GSSG (final
concentration 1 mM). The GR activity was assayed by monitoring the decrease in absorbance at 340
nm during the initial 3 min. The GR activity was expressed as percentage of the control.
4.7. Imaging TrxR activity in HL-60 cells by TRFS-green
We employed TRFS-green, a specific fluorescence probe of TrxR, to image TrxR activity in
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intact cells according to our published protocols [42]. Briefly, HL-60 cells (1×106) were incubated
with indicated concentrations of 3 in 12-well plates for 8 h, then followed by further incubation with
TRFS-green (10 µM) for 4h at 37 °C. The fluorescence and bright field images were acquired.
4.8. Assay of TrxR activity in cell lysates by the endpoint insulin reduction assay
HL-60 cells (70-80% confluence) were incubated with varying concentrations of 3 for 24 h.
They were harvested, and washed twice with phosphate-buffered saline (PBS). Total cellular proteins
were extracted by RIPA buffer (50 mM Tris-HCl pH 7.5, 0.5% deoxycholate, 2 mM EDTA, 0.1%
SDS, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF and 1 mM Na3VO4) for 30 min on ice, and
were quantified using the Bradford procedure. TrxR activity in cell lysates was assayed by the
endpoint insulin reduction assay according to our published protocols [41].
4.9. Measurement of ROS production
HL-60 cells (5×105) were plated in 12-well plates and were treated with the indicated
concentrations of 3 for 1 h. The ROS indicator DHE (10 µM) or DCFH-DA (10 µM) in fresh
FBS-free medium was added to each well, and further incubated for 30 min at 37 °C. The cells were
viewed and images were acquired.
4.10. Measurement of intracellular thiols
After treatment of HL-60 cells (2×106) with increasing concentrations of 3 for 24 h in 100 mm
dishes, the cells were collected, and washed twice with PBS. Total cellular proteins were extracted
by RIPA buffer, and were quantified using the Bradford procedure. Total cellular thiols were
measured by DTNB-titration. Briefly, cell lysate (10 µL) was added to cuvettes containing DTNB (1
mM in 90 µL of 6 M guanidine hydrochloride, pH 8.0). After incubation for 5 min at room
temperature, the absorbance at 412 nm was read on a microplate reader. Total thiols were calculated
from a calibration curve using GSH as the standard.
4.11. Measurement of cellular glutathiones
Measurement and quantification of the total glutathione and GSSG was based on the enzymatic
recycling method according to our previously published [41]. Cells (2×106) were treated with the
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indicated concentrations of 3 for 24 h in 100 mm dishes, and were resuspended using ice-cold
extraction buffer containing 0.1% Triton X-100 and 0.6% sulfosalicyclic acid in 0.1 M potassium
phosphate buffer with 5 mM EDTA, pH 7.5 (KPE buffer). After sonication of the suspension in ice
water for 2-3 min with vortexing every 30 s, the solution was centrifuged at 3000 g for 4 min at 4 °C,
and the supernatant was immediately collected. To assay the total glutathione, a solution (120 µl)
containing 1.66 units/ml GR and 0.33 mg/ml DTNB was added to each sample (20 µl). Then
NADPH (60 µl of 0.66 mg/ml) was added and the absorbance at 412 nm was immediately readed
every 10 s for 2 min. GSSG was determined after GSH derivatization by 2-vinylpyridine. Briefly,
two microliters of 2-vinylpyridine was added to 100 µl of cell supernatant and mixed, then the
reaction was allowed to take place for 1 h at room temperature in a fume hood. Finally, six
microliters of triethanolamine was added to the supernatant and the solution was mixed. Assay of
GSSG was performed as described above for total glutathione. The amount of GSSG was subtracted
from the total glutathione to give the GSH content.
4.12. Western blot analysis
For western blot analysis, total cellular proteins were extracted by RIPA buffer, and were
quantified using the Bradford procedure. Equal amounts of protein in each lysate sample were
separated by SDS-PAGE (12% gel, 40 µg per lane) under reducing conditions and then transferred to
poly-vinylidene difluoride (PVDF) membranes (Millipore, USA). The blots were blocked with 5%
non-fat milk in TBST at room temperature for 2 h, then membranes were incubated with specific
primary antibodies in 5% non-fat milk at 4 °C overnight. Following three washes with TBST, the
membranes were incubated with the peroxidase conjugated secondary antibodies for 1 h at room
temperature. The immunoreactive bands were visualized by using an enhanced chemiluminescence
kit from GE Healthcare.
4.13. Apoptosis assays
4.13.1. Assessment of caspase 3 activity
After treatment of HL-60 cells (2×106) with varying concentrations of 3 for 24 h in 100 mm
dishes, the cells were collected, and washed twice with PBS. Total cellular proteins were extracted
by RIPA buffer, and were quantified using the Bradford procedure. A cell extract containing 50 µg of
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total proteins was incubated with the assay mixtures (50 mM Hepes, 0.1% CHAPS, 2 mM EDTA, 5%
glycerol, 10 mM DTT, 0.2 mM Ac-DEVD-pNA, pH 7.5) for 2 h at 37 °C in a final volume of 100 µl.
The absorbance at 405 nm was readed by using a microplate reader. Controls were treated with the
same amounts of DMSO alone and the caspase 3 activity was expressed as the percentage of the
control.
4.13.2. Annexin V/PI staining
HL-60 cells (1×106) were incubated with 0.5, 1, 3 and 5 µM 3 for 24 h in 6-well plates. The
cells were harvested and washed twice with PBS. The apoptotic cells, necrotic cells and live cells
were identified by the PI double and Annexin V-FITC staining assay according to the manufacturer’s
instructions. After staining, the cells were determined by a FACSCantoTM flow cytometer (BD
Biosciences, USA), and the data were analyzed with the CellQuest software.
4.13.3. Hoechst 33342 staining
HL-60 cells (1×105) were plated in 12-well plates and incubated with 0.5, 1, 3 or 5 µM 3 for 24
h. Hoechst 33342 was added to reach a final concentration of 5 µg/ml and further incubated for 20
minutes. The cells were viewed and photographed.
4.14. Molecular docking simulation
A covalent docking was performed in the program Schrödinger Suite (Schrödinger, LLC: New
York, NY, 2015) to investigate the binding mode of compound 3 and the enzyme. A rat TrxR1
structure (PDB code 3EAN [48], Chain A and B) was obtained from the Protein Data Bank and
further prepared in the Protein Preparation Wizard module. The residue Sec498 in chain A was
selected as the reactive residue involved in the Michael addition and also set as the centroid of the
docking pocket. The default parameters were used in the docking simulation.
4.15. Statistics
All data were presented as mean ± SE from 3-5 different experiments. The differences between
sets of data were assessed by the Students t-test. Compare means between control and treatment
groups were performed by using one-way analysis of variance (ANOVA). A p value <0.05 was used
as the criterion for statistical significance.
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Acknowledgements
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The financial supports from Natural Science Foundation of China (21572093, 21778028) and
the Fundamental Research Funds for the Central Universities (lzujbky-2016-49) are greatly
acknowledged. The authors also appreciate Prof. Arne Holmgren (Karolinska Institute, Sweden) for
the recombinant rat TrxR1 and Prof. Constantinos Koumenis (University of Pennsylvania) for cells
and shRNA plasmids.
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Appendix A. Supplementary data
The following is the supplementary data related to this article:
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Conflicts of interest
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The authors confirm that there are no conflicts of interest.
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Scheme 1 Synthesis of compounds 1-16. (a) 1) AlCl3/NaCl, 165 °C for 4 h; 2) 10% HCl; (b) R-NH2,
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EtOH, rt.
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Table 1 Cytotoxicity of all compounds (1-16) against human promyelocytic leukemia HL-60 cells.
Compound
R substituents
Cell viability (%)*
—
23.08 ± 0.12
0
1
2
3
4
5
6
7
8
9
—
—
0
NHCH3
62.96 ± 1.69
56.39 ± 1.52
59.38 ± 0.66
59.21 ± 2.68
61.10 ± 1.43
50.28 ± 0.36
NHCH2CH3
NH(CH2)2CH3
NH(CH2)3CH3
NH(CH2)7CH3
NH(CH2)2OH
57.04 ± 2.16
54.26 ± 1.92
10
11
59.72 ± 2.98
54.58 ± 1.65
54.85 ± 1.28
50.13 ± 3.29
97.23 ± 2.21
12
13
14
15
16
670
* Cells were treated with the tested compounds (20 µM) for 48 h and the cell viability was
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determined by the MTT assay.
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Fig. 1 Cytotoxicity of compounds 2 and 3 in cells. (A) Time-dependent cytotoxicity of 2 or 3
towards HL-60 cells. The HL-60 cells were incubated with the varying concentrations of 2 or 3 for
the indicated times. Cell viability was assessed by the MTT method. (B) Cytotoxicity of 3 towards
HL-60, A549, HepG 2, and HeLa cells. The cells were incubated with the indicated concentrations of
3 for 48 h and the cell viability was assessed by the MTT method. (C) Cytotoxicity of 3 towards
HL-60 for 48 h. Cell viability was determined by the trypan blue exclusion assay. (D) Cytotoxicity of
3 towards L02 and HEK 293T cells in comparison with HL-60 cells. Cells were treated with the
compound for 48 h, and the cell viability was assessed by the MTT method. Data are expressed as
mean ± SE from triplicates. **, P< 0.01 vs. the control groups in (A) & (C); **, P< 0.01 vs. the
normal cells in (D).
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Fig. 2 Targeting the Sec residue of TrxR by 3. (A) Inhibition of purified TrxR, U498C TrxR and GR
by 3. NADPH-reduced recombinant rat TrxR1 (WT TrxR1), U498C TrxR1, and GR were incubated
with the indicated concentrations of 3 for 1 h at room temperature, and the enzymes’ activity was
determined and expressed as the percentage of the control. (B) Molecular docking of compound 3
with TrxR1 was carried out using the covalent docking protocol in the program Schrödinger Suite
(Schrödinger, LLC: New York, NY, 2015). The docking score for the non-bonding interaction was
-5.81 kcal/mol. Two subunits of TrxR1 were represented by green and cyan cartoons respectively.
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Fig. 3 Involvement of TrxR for the cellular action of 3. (A) Imaging the cellular TrxR activity in live
HL-60 cells by TRFS-green. After treatment of HL-60 cells with varying concentrations of 3 for 12 h,
the TrxR activity was stained by the TrxR probe TRFS-green. The bright filed pictures (top panel)
and the fluorescence pictures (bottom panel) were acquired. Scale bars: 20 µm. (B) Inhibition of
TrxR activity in HL-60 cells by 3. After the cells were treated with the indicated concentrations of 3
for 24 h, the TrxR activity in cells was determined by the end point insulin reduction assay. (C) No
obvious changes of TrxR1 protein levels in HL-60 cells treated with 3. Cells were treated with the
indicated concentrations of 3 for 24 h, and the cell extracts were assessed by Western blots. (D)
Overexpression of TrxR1 decreases the cytotoxicity of 3. The HEK-IRES and HEK-TrxR1 cells were
incubated with the various concentrations of 3 for 48 h, and the cell viability was assayed by the
MTT method. (E) Knockdown of TrxR1 increases the cytotoxicity of 3. The HeLa-shTrxR1and
HeLa-shNT cells were incubated with the indicated concentrations of 3 for 48 h, and the cell viability
was assayed by the MTT method. Data are expressed as mean ± SE of three experiments. **, P< 0.01
vs. the control groups in (B); **, P< 0.01 vs. the HEK-IRES cells in (D) and HeLa-shNT cells in (E).
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