Synthesis of xanthohumol analogues and discovery of potent thioredoxin reductase inhibitor as potential anticancer agent

Article abstract of DOI:10.1021/jm5016507

The selenoprotein thioredoxin reductases (TrxRs) are attractive targets for anticancer drugs development. Xanthohumol (Xn), a naturally occurring polyphenol chalcone from hops, has received increasing attention because of its multiple pharmacological activities. We synthesized Xn and its 43 analogues and discovered that compound 13n displayed the highest cytotoxicity toward HeLa cells (IC₅₀ = 1.4 μM). Structure-activity relationship study indicates that the prenyl group is not necessary for cytotoxicity, and introducing electron-withdrawing group, especially on the meta-position, is favored. In addition, methylation of the phenoxyl groups generally improves the potency. Mechanistic study revealed that 13n selectively inhibits TrxR and induces reactive oxygen species and apoptosis in HeLa cells. Cells overexpressing TrxR are resistant to 13n insult, while knockdown of TrxR sensitizes cells to 13n treatment, highlighting the physiological significance of targeting TrxR by 13n. The clarification of the structural determinants for the potency would guide the design of novel potent molecules for future development.

Full text of DOI:10.1021/jm5016507

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Article
Synthesis of Xanthohumol Analogues and Discovery of Potent
Thioredoxin Reductase Inhibitor as Potential Anticancer Agent
Baoxin Zhang, Dongzhu Duan, Chunpo Ge, Juan Yao, Yaping Liu, Xinming Li, and Jianguo Fang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5016507 • Publication Date (Web): 28 Jan 2015
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Synthesis of Xanthohumol Analogues and Discovery of Potent Thioredoxin
Reductase Inhibitor as Potential Anticancer Agent
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Baoxin Zhang, Dongzhu Duan, Chunpo Ge, Juan Yao, Yaping Liu, Xinming Li, Jianguo Fang*
State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical
Engineering, Lanzhou University, Lanzhou 730000, China
*Corresponding author, Eꢀmail: fangjg@lzu.edu.cn (J. Fang); Fax: +86 931 8915557.
ABSTRACT
The selenoprotein thioredoxin reductases (TrxRs) are attractive targets for anticancer drugs
development. Xanthohumol (Xn), a naturally occurring polyphenol chalcone from hops, has
received increasing attention due to its multiple pharmacological activities. We synthesized Xn
and its 43 analogues, and discovered compound 13n displayed the highest cytotoxicity towards
HeLa cells (IC50=1.4 ꢀM). Structureꢀactivity relationship study indicates that the prenyl group is
not necessary for the cytotoxicity, and introducing electronꢀwithdrawing group, especially on the
meta-position, is favored. In addition, methylation of the phenoxyl groups generally improves the
potency. Mechanistic study revealed that 13n selectively inhibits TrxR, induces reactive oxygen
species and apoptosis in HeLa cells. Cells overexpressing TrxR are resistant to 13n insult, while
knockdown of TrxR sensitizes cells to 13n treatment, highlighting the physiological significance
of targeting TrxR by 13n. The clarification of the structural determinants for the potency would
guide the design of novel potent molecules for future development.
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INTRODUCTION
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The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and
NADPH, is a highly conserved, ubiquitous network in all cells, and plays crucial roles in the
redox regulation of numerous cellular signaling pathways involved in cell survival and
proliferation.1ꢀ3 Reduced Trx carries on the principal function of the system via a reversible
thiolꢀdisulfide exchange reaction during transfer of reducing equivalents to various cellular targets
to generate an oxidized form of Trx, which is in turn recycled to a reduced state by taking
electrons from NADPH under TrxR catalysis. As TrxR is the only known physiological enzyme
to catalyze the reduction of oxidized Trx, the function of the thioredoxin system is strictly
controlled by the activity of TrxR. Mammalian TrxRs, compared to those from bacteria, are large
seleniumꢀcontaining proteins with a penultimate selenocysteine (Sec) in the Cꢀterminal active
site.4, 5 This flexible Cꢀterminal extension enables TrxR to extend the electron transport chain
from the catalytic disulfide to the enzyme surface. Mammalian TrxRs have a broad range of
substrates and are easily inactivated by various alkylating agents6, 7 presumably due to the
presence and easy accessibility of the high reactive Sec residue in the reduced form of the
enzymes. In recent years, accumulating evidence supports that TrxR is a promising target for
development of novel anticancer agents as the thioredoxin system is often overexpressed in many
tumors,8, 9 and this overexpression confers drug resistance in cancer chemotherapy.10 The
physiological importance of TrxR for tumor progression was demonstrated by Hatfield et al.,
showing that knockdown of TrxR prevents tumor formation, and inhibits selfꢀsufficient growth
and DNA replication of cancer cells.11, 12 Thus, the past years have witnessed a great endeavor in
discovering and developing a variety of TrxRꢀtargeted small molecules as potential therapeutic
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agents.6, 13ꢀ22
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Natural products and their derivatives are invaluable fountain of therapeutic agents, and have
driven pharmaceutical discovery over the past century. The diversity of natural products continue
to provide a unique source of bioactive lead compounds for drug development.23, 24 It is roughly
estimated that half of modern marketed drugs are natural products or their derivatives. In the case
of anticancer and antiꢀinfective agents, this proportion is even higher.23, 25 As essence of the drug
discovery progress, generation of novel and structurally diverse molecules with potential
biological properties has been actively pursued. Chalcones, also known as α, βꢀunsaturated
ketones, form a central core of various natural products, such as flavokawain, butein, licochalcone,
xanthoangelol, xanthohumol, derricin, cardamonin, isoliquiritigenin, isosalipurposide, and
naringenin. The chalcones family has been documented with diverse biological function,
including immunomodulation, antiꢀinflammation, anticancer, and antidiabetic activity, and some
of them have been approved for clinical application or tested in humans.26, 27 Xanthohumol, a
structurally simple prenylated chalcone, is the major prenylflavonoid that is extracted from the
hop plant, Humulus lupulus, which is widely used in beers and a few types of soft drinks, such as
Julmust and Malta. The presence of high concentration of Xn in beers has been suggested to link
to the epidemiological observation of the beneficial effect of regular beer drinking.28
Xanthohumol has been characterized as a ‘broadspectrum’ anticancer agent,29 and some putative
mechanisms underlying the effect of Xn have been reported, including generation of ROS,30ꢀ32
downregulation of antiꢀapoptotic proteins,33ꢀ36 upregulation of p53,37, 38 and induction of phase II
enzymes.39, 40 Despite the wellꢀdocumented anticancer activity of Xn, its efficacy is moderate, and
hence it is of demand to improve the potency of Xn for future development.
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As our continuous interests in discovering and developing small molecule regulators of
cellular redox system as potential therapeutic agents,6, 15, 41ꢀ47 we synthesized a series of Xn
analogues, and evaluated their cytotoxicity in different types of cancer cells. In the cultured HeLa
cells, compound 13n displayed the greatest potency with an IC50 value of 1.4 ꢀM, more than
30ꢀfold increase compared to the lead compound Xn. Further mechanistic studies disclose that
13n may selectively inhibit TrxR by primarily targeting the Sec residue in the antioxidant enzyme,
leading to the accumulation of ROS. As a consequence, 13n elicits oxidative stress, and
eventually induces apoptosis in HeLa cells. Importantly, overexpression of the functional TrxR in
cells alleviates the cytotoxicity of 13n, while knockdown of the enzyme augments the cytotoxicity,
supporting the physiological significance of targeting TrxR by 13n.
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RESULTS AND DISCUSSION
Chemistry. Analysis of the chemical structure of Xn reveals it belongs to the chalcones
family, the structure of which could be easily constructed by the baseꢀcatalyzed Claisen-Schmidt
condensation of an aldehyde with a ketone in a polar solvent like ethanol or methanol. Numerous
bases have been applied in the condensation reaction, including NaOH, KOH, Ba(OH)2, and
LiHMDS, which were summarized in the recent publication.48 We employed KOH as a catalyst in
anhydrous methanol for the construction of the skeleton of all chalcone derivatives used in the
present study (Schemes 1ꢀ3).49, 50 In brief, the different substituted acetophenone was dissolved in
anhydrous methanol followed by addition of a catalytic amount of KOH. The reaction mixture
was stirred at room temperature for an appropriate time followed by the addition of diverse
substituted benzaldehyde. The reaction was carried out at room temperature until completion, and
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the corresponding chalcone derivative (8aꢀ8n, 11aꢀ11n, and 13aꢀ13q) was isolated by
crystallization or by silica gel flash chromatography. For the synthesis of Xn and its prenylated
chalcone derivatives, insertion of a prenyl group onto the aryl ring was achieved by a paraꢀClaisen
rearrangement after using a Mitsunobu reaction to establish the key prenyl ether precursor (4). A
Claisen–Schmidt condensation was deployed to construct the chalcone scaffold followed by
removal of methoxymethyl (MOM) protecting groups under acidic conditions that were optimized
to prevent concomitant cyclization to the flavone. All chalcone derivatives were fully
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characterized by H, 13C NMR, and MS. The purity of all chalcones was determined by HPLC
analysis, and was greater than 95%.
Cytotoxicity of Xn and its analogues. Initially, we adopted the MTT assay to screen the
cytotoxicity of all compounds (8a-8m, 11a-11n, and 13a-13q) towards three different types of
cancer cell lines. Under this condition, the concentrations that inhibit the cell proliferation to 50%
of the control (IC50 values) were summarized in Tables 1ꢀ3. Several compounds showed
promising cytotoxicity, such as 8l, 11n, 13m, 13n, and 13o. Next, we chose these five compounds
for further followꢀup studies. As listed in Table 4, compound 13n is the most potent one among all
the tested compounds with the highest potency towards HeLa cells (IC50=1.4 ꢀM). To confirm the
results from the MTT assay, we also employed the trypan blue exclusion assay to determine the
cell viability upon treatment of HeLa cells with varying concentrations of 13n, which gives an
IC50 value of 1.7 ꢀM, quite consistent with the value from the MTT assay. Compared to Xn
(IC50>40 ꢀM for HeLa cells), the cytotoxicity of 13n was greatly improved.
Inhibition of TrxR by 13n in vitro and in cells. Since 13n displays the highest cytotoxicity
to HeLa cells, we then asked the possible cellular target of the compound. The core feature of
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chalcones is that they contain an α, βꢀunsaturated keton moiety, a key structure for many reported
TrxR inhibitors.43ꢀ45, 51ꢀ54 Thus we speculated that 13n might be a novel inhibitor of TrxR. As
shown in Fig. 1A, 13n gave a clear inhibition of TrxR with an IC50 value around 12.8 ꢀM (line
with closed squares). As many TrxR inhibitors have been demonstrated to target the Sec residue
in the enzyme, we further determined the inhibition of 13n towards the mutant enzyme, U498C
TrxR, where the Sec498 was replaced by Cys. Compared to the wild type TrxR, 13n gave very
weak inhibition to U498C TrxR (line with open circles), suggesting that targeting the Sec residue
is involved in the inhibition of TrxR. Glutathione reductase (GR) is a homologous of TrxR with
similar overall structure. Again, 13n had marginal effect on the enzyme activity (line with closed
triangles). We also determined the possible inhibition of glutathione peroxidase (GPx), a
Secꢀcontaining enzyme, by 13n, and no apparent inhibition of the GPx was observed (line with
open inverted triangles). Taken together, 13n selectively targets the Sec residue in TrxR to inhibit
its activity in vitro. We also evaluated the inhibition of TrxR by Xn, 8l, 11n, 13m, and 13o. As
shown in Fig. S1 in the Supporting Information (SI), all the tested compounds are inhibitors of
TrxR, while compound 13n gives the most potent inhibition. Next, we determined the TrxR
activity in HeLa cells after 13n treatment. Two different assays, i. e., the classic Trxꢀmediated
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insulin reduction assay developed by Holmgren55
and the fluorogenic probe
(TRFSꢀgreen)ꢀbased live cell imaging assay developed by our group,41 were employed. Both
assays gave consistent results and displayed clear concentrationꢀdependent inhibition of TrxR by
13n in HeLa cells (Fig. 1B & C). The IC50 value of 3.5 ꢀM could be obtained from the
Trxꢀmediated insulin reduction assay. Compound to the lead compound Xn (IC50>40 ꢀM, Fig.
1B), the inhibition of cellular TrxR by 13n was much improved. One of the functions of the
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thioredoxin system is to prevent the oxidation of cellular thiols, especially the protein thiols. As a
consequence of inhibition of TrxR by 13n, the cellular thiols decreased remarkably after 13n
treatment (Fig. S2 in SI).
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Induction of ROS in cells by 13n. One of the outstanding functions of the Trx system is to
maintain the intracellular redox homeostasis and defend against oxidative stress. Besides its direct
counteraction of ROS, TrxR is the only known enzyme in maintaining reduced Trx pools for
ribonucleotide reductase in DNA synthesis,56 and many antioxidant enzymes such as
peroxiredoxins57 and methionine sulfoxide reductases58 under physiological conditions. Inhibition
of TrxR would be expected to disturb the cellular redox balance and cause accumulation of ROS
in cells. DCFHꢀDA is a wellꢀestablished probe to detect intracellular production of ROS. After
uptaken by cells, DCFHꢀDA is hydrolyzed by cellular esterases to dichlorodihydrofluorescin
(DCFH), which is trapped within the cell. The nonfluorescent DCFH is then oxidized to
fluorescent dichlorofluorescin by action of cellular ROS. As shown in Fig. 2, the ROS level in
HeLa cells is undetectable under basal conditions. Upon treatment of the cells with 13n, a
remarkable elevation of the ROS production was observed both in a short time treatment (Fig. 2A)
and a long time treatment (Fig. 2B). Under the same experimental conditions, Xn alone gives no
detectable fluorescence signal.
Physiological significance of targeting TrxR by 13n in HeLa cells. As we have
demonstrated the selective interaction of 13n with TrxR in vitro and potent inhibition of TrxR in
HeLa cells, we then asked the physiological relevance of the cytotoxicity and TrxR inhibition by
13n. Pretreatment of HeLa cells with Nꢀacetylcysteine (NAC), a thiol antioxidant and GSH
synthesis precursor in cells, confers cytoprotection against 13nꢀinduced cell death (Fig. 3A).
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Subsequently, we determined the effect of GSH on the cytotoxicity of 13n. Consistent with the
protection by NAC, inhibition of cellular GSH synthesis by BSO significantly enhances the
cytotoxicity of 13n (Fig. 3B). Under our experimental conditions, pretreatment of HeLa cells with
50 ꢀM BSO for 24 h decreases the cellular GSH level to ~30% of the control (Fig. S3 in SI). GSH
is a pivotal component of the glutathione network, which is another redox regulation system in
cells besides the thioredoxin system, and also acts as a backup of the thioredoxin system.59
Depletion of GSH sensitizing the cells to 13n supports the involvement of the thioredoxin system
in the biological action of 13n. To disclose the involvement of TrxR in the cytotoxicity of 13n, we
turned to compare the sensitivity of HEK cells stably overexpressing TrxR1 (HEKꢀTrxR1) and
the cells that stably transfected with a vector (HEKꢀIRES) towards 13n treatment. The TrxR1
expression level in HEKꢀTrxR1 cells are ~3ꢀꢀfold higher than that in HEKꢀIRES cells under our
experimental conditions.43, 44 As shown in Fig. 4A, 13n displays remarkably higher cytotoxicity to
HEKꢀIRES cells than HEKꢀTrxR1 cells. To further address the physiological significance of TrxR
in 13nꢀinduced cytotoxicity, we generated a cell line stably knocking down TrxR1 expression
(HeLaꢀshTrxR1 cells) by transfection of shRNA specifically targeting the enzyme. The TrxR1
expression level in HeLaꢀshTrxR1 cells is only about 20% of that in HeLaꢀshNT cells, which were
transfected with a nonꢀtargeting shRNA. Importantly, 13n shows significantly elevating
cytotoxicity to HeLaꢀshTrxR1 cells (Fig. 4B). The efficiency of knockdown and overexpression
of TrxR1 in cells was fully evaluated in our recent publications.43, 44 Collectively, our results
strongly supported that the biological action of 13n in HeLa cells is dependent on its interaction
with TrxR.
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Induction of apoptosis by 13n. It has been reported that Xn has ability to activate apoptotic
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signaling in numerous type of malignant cells.31, 32, 34, 38, 39, 60 Herein, we also demonstrated that
13n kills HeLa cells predominantly through the induction of apoptosis. When HeLa cells were
incubated with 2 ꢀM or 5 ꢀM of 13n followed by Hoechst staining, an increasing number of cells
displayed condensed nuclei, a characteristic morphology of cells undergoing apoptosis (Fig. 5A).
Caspase 3 is a crucial component of the apoptotic machinery in different cell types, and the
activation of caspase 3 is a central event in the process of apoptosis. Thus, we next determined the
activity of caspase 3 after the cells exposure to 13n. As shown in Fig. 5B, the caspase 3 activity in
HeLa cells was strikingly increased after treatment with varying concentrations of 13n. Further
evidence from the annexinꢀV–fluoresceinꢀ5ꢀisothiocyanate (FITC)/propidium iodide (PI) double
staining assay confirms that 13n almost exclusively triggers apoptotic cell death in a
doseꢀdependent manner (Fig. 5C). Taken together, our data indicated that 13n mainly induces
apoptotic cell death in HeLa cells.
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TrxR is a pivotal component of the thioredoxin system, which is essential for maintaining the
cellular redox balance, and is involved in regulation of a wide range of redox signaling. The
malfunction of the system has been suggested to link to various diseases.2 Inhibition of TrxR
reduces the available pool of reduced Trx, leading to a decrease in the activity of many enzyme
systems that require Trx as an electron donor,1, 61 such as ribonucleotide reductase, peroxiredoxins,
and methionine sulfoxide reductases. Furthermore, the reduced Trx, but not the oxidized one,
directly interacts with a variety of apoptosisꢀrelated proteins, such as ASK1,62 procaspase 3,63
APꢀ164 and NFꢀκB,65 to suppress apoptosis, and thus TrxR inhibition promotes apoptosis.
Inhibition of TrxR is more detrimental to cancer cells as cancer cells generally require constant
DNA synthesis due to their fast proliferation, demand high antioxidant enzymes activity due to
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their intrinsic elevated oxidative stress,66 and display abrogation or resistance of apoptosis due to
misregulated signaling pathways.67 Targeting TrxR is a promising strategy to develop novel
therapeutic agents.
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Chalcones (1,3ꢀdiphenylpropenꢀ1ꢀones) are naturally occurring compounds belonging to the
flavonoid family and display a number of pharmacological functions.26, 27 The presence of a
double bond in conjugation with the carbonyl functionality (α, βꢀunsaturated ketone) is believed
to be responsible for the biological activities of chalcones. Compounds with this structure in
chemical libraries are usually considered as PAINS (Pan Assay Interference Compounds) which
have the potential to cause misleading results in highꢀthroughput screening.68 The α,
βꢀunsaturated ketone structure is one of the wellꢀdocumented pharmacophores that targets the
selenoprotein TrxR, which is the basis of the current work. All the synthesized compounds equally
contain such structure, and the promising compound (13n) from the initial evaluation was fully
determined to demonstrate its specific interaction with TrxR in the followꢀup studies, thus
precluding the false positive incidence. We have modified the substitute groups of Xn to furnish a
series of Xn analogues. Based on the cytotoxicity screening results (Tables 1ꢀ3), preliminary
structureꢀactivity relationship could be drawn: 1) The prenyl group is not necessary for the activity,
as 11m, 11n, 13h, and 13n are highly cytotoxic; 2) Molecules bearing electronꢀwithdrawing
groups (EWG), such as 8l, 11m, and 13lꢀp, generally have better activity; 3) The EWG located at
the metaꢀposition (11n and 13n) generally leads to higher activity than those at orthꢀ or
paraꢀposition (11l and 13l-p); 4) Methylation of the hydroxyl groups on the benzene ring close to
the carbonyl group commonly improves the potency, such as 11j vs 13e, 11k vs 13o, 11l vs 13l,
11m vs 13m and 11n vs 13n. The dispensability of the prenyl group for the activity would greatly
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facilitate preparing more Xn analogues, as the introduction of the prenyl group is the most tedious
step in synthesizing such kind of compounds (Step d in Scheme 1). We previously reported that
several natural products bearing α, βꢀunsaturated ketone structure, such as curcumin,45, 54
gambogic acid43 and shikonin,44 are potent TrxR inhibitors. In analogy to the inhibition of TrxR
by those molecules, we reasoned that binding of 13n to the enzyme might be the molecular basis
for the inhibition of TrxR. The presence of the prenyl group makes the molecule steric and
lipophilic, which is unfavorable for interacting with TrxR. Increasing the potency by removing the
prenyl group would be another advantage for developing more potent TrxR inhibitors, as the steps
of introducing the prenyl group are the most sluggish processes (Scheme 1, steps c and d) in
synthesizing Xn analogues. Introducing the EWG to the molecules enhances the potency could be
due to that the EWG makes the double bond more electronꢀdeficient, thus facilitating the
nucleophilic addition from TrxR. The potency of inhibition of TrxR by 13n is moderate as several
known TrxR inhibitors have lower IC50 values than 13n does.14, 15, 22, 43, 44 However, a great
improvement has been achieved if comparing the potency of TrxR inhibition and cytotoxicity by
13n to the lead natural product Xn. In this work, we kept the core chalcone structure, and mainly
modified the benzene ring attached to the double bond. We are currently undergoing the structure
modifications of the other benzene ring attached to the carbonyl group. We expected these further
modifications would yield more potent TrxR inhibitors.
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The specificity of 13nꢀTrxR interaction was demonstrated by the following evidence. Firstly,
we compared the in vitro inhibition of 13n to TrxR, U498C TrxR, GR (a homologous to TrxR)
and GPx (a Secꢀcontaining enzyme) (Fig. 1). Single mutation of Sec to Cys sharply decreased the
enzyme sensitivity to 13n, indicating that the Sec residue in TrxR is a primary target for 13n. The
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structure of GR is quite closely related to TrxR, however, no apparent inhibition of GR was
observed under our experimental conditions. Furthermore, 13n displays negligible effect to GPx,
which contains a buried Sec residue. The selective inhibition of WT TrxR, but not U498C TrxR,
GR or GPx, suggests a specific interaction of 13n and TrxR. This selectivity could be due to the
binding of 13n to the Sec residue as the Sec presents on the surface of the enzyme, and is more
reactive than Cys at physiological conditions. Secondly, we provided evidence in cellular context
to support the unique role of TrxR in 13n’s biological action. HEK cells stably overexpressing
TrxR (HEKꢀTrxR1 cells) show less sensitivity to 13n treatment compared to the cells only
transfected with the vector (HEKꢀIRES) (Fig. 4A). More physiologically relevant evidence that
genetic knockdown of TrxR elevates the compound’s cytotoxicity further supports that TrxR is
critically involved in the biological function of 13n (Fig. 4B). The glutathione system and
thioredoxin system are two major networks that work independently but with some overlaps in
regulating the intracellular redox balance. Recent studies suggested that the glutathione system
can act as a backup of the thioredoxin system.59 Our observations that deletion of cellular GSH by
BSO enhances 13n’s cytotoxicity, while elevation of GSH by NAC alleviates the cytotoxicity (Fig.
3), also support that TrxR is involved in the biological action of 13n. Taken together, our data
suggest that 13n targets TrxR in HeLa cells with high specificity.
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Apoptosis is a consequence of a series of precisely controlled process that occurs in
physiological and pathological conditions, and is frequently altered in cancer cells. Two
wellꢀknown apoptosis signaling pathways, i.e., the extrinsic cell surface receptor pathway and the
intrinsic mitochondrial pathway, converge on the caspases activation. Evading apoptosis, which
represents a major causative factor in the development and progression of cancer, is a hallmark in
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diverse malignant cells arising from a complex interplay of genetic aberrations and misregulated
pathways.67, 69 Our results indicated that compound 13n shows potent cytotoxicity to HeLa cells
predominantly through induction of apoptosis (Fig. 5), thus potentiating the further development
of 13n for treatment of tumors. ROS and mitochondria play an important role in apoptosis process.
Induction of ROS usually accompanies with the inhibition of TrxR.15, 43ꢀ45 The elevated ROS level
impairs the mitochondria function, leading to release of cytochrome c to the cytocol, and
subsequently activates the caspases family. As we have observed that 13n could promote
accumulation of ROS (Fig. 2) and induction of apoptosis in HeLa cells (Fig. 5), it is most likely
that 13n prompts apoptosis through the oxidative stressꢀinduced intrinsic mitochondrial pathway.
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CONCLUSIONS
In summary, we have synthesized Xn and its 43 analogues. After cytotoxicity screening, 13n was
identified as the most cytotoxic compound towards HeLa cells with an IC50 value of 1.4 ꢀM,
which is more than 30ꢀfold increase of potency than the lead natural product Xn. Preliminary
structureꢀactivity relationship study indicates that the prenyl group is not necessary for the
cytotoxicity, and introducing EWG, especially on the meta-position, into molecules is favored for
the activity. In addition, methylation of the hydroxyl groups on the benzene ring generally
improves the potency. The clarification of the structural determinants for the potency would guide
the design of novel potent molecules for future development. The followꢀup studies discovered
that 13n could selectively inhibit the redox enzyme TrxR in vitro, and effectively targets TrxR in
HeLa cells. Further mechanistic investigation revealed that 13n could induce ROS production and
promote oxidative stressꢀmediated apoptosis in HeLa cells. The observation that cells
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overexpressing TrxR are more resistant to 13n insult, while knockdown of TrxR sensitizes cells to
13n treatment accentuates the physiological significance of targeting TrxR by 13n.
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EXPERIMENTAL PROCEDURES
Materials. The recombinant rat TrxR1 was essentially prepared as described70, and is a gift
from Prof. Arne Holmgren at Karolinska Institute, Sweden. The recombinant U498C TrxR mutant
(Sec→Cys) and truncated TrxR (TꢀTrxR) were produced as described.15, 71 The U498C TrxR is
active in DTNB assay, and the activity of the mutant enzyme is ~10% of that of the recombinant
wild type enzyme. Proteins were pure as judged by Coomassieꢀstained SDSꢀpolyacrylamide gel
electrophoresis (PAGE). E. coli Trx was purchased from IMCO (Stockholm, Sweden,
www.imcocorp.se). RPMI 1640 Medium, Dulbecco's modified Eagle’s medium (DMEM), G418,
NAC,
bovine
insulin,
Lꢀbuthionineꢀ(S,R)ꢀsulfoximine
(BSO),
NꢀacetylꢀAspꢀGluꢀValꢀAspꢀpꢀnitroanilide (AcꢀDEVDꢀpNA), reduced and oxidized glutathione
(GSH and GSSG), dimethyl sulfoxide (DMSO), yeast glutathione reductase (GR), glutathione
peroxidase
(GPx),
superoxide
dismutase
(CHAPS),
(SOD),
3ꢀ[(3ꢀCholamidopropyl)dimethylammonio]ꢀ1ꢀpropanesulfonate
DLꢀdithiothreitol
(DTT), puromycin and 2’,7’ꢀdichlorfluorescein diacetate (DCFHꢀDA) were obtained from
SigmaꢀAldrich (St. Louis, USA). Cytochrome c and NiꢀNTAꢀSefinose were obtained from Sangon
Biotech (Shanghai, China). NADPH was obtained from Roche (Mannheim, Germany). Fetal
bovine
serum
(FBS)
was
obtained
from
Sijiqing
(Hangzhou,
China).
3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide
(MTT), penicillin and
streptomycin were obtained from Amresco (Solon, USA). Bovine serum albumin (BSA),
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phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate (Na3VO4), ethylene
diaminetetraacetic acid (EDTA) and 5,5’ꢀdithiobisꢀ2ꢀnitrobenzoic acid (DTNB) were obtained
from J&K Scientific (Beijing, China). Melting points (mp) were determined on a FisherꢀJohns
melting apparatus and were uncorrected.1H NMR13C NMR spectra were recorded with a Bruker
AMX spectrometer at 400 and 100 MHz, respectively, using TMS as the internal standard
(chemical shifts are given in δ values, J is given in Hz). Mass spectra were obtained using a
HewlettꢀPackard 5988A spectrometer. The purity of final compounds was assessed by HPLC and
was found to be higher than 95%. 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.
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Compounds Purity Analysis. All final compounds were analyzed by HPLC to determine
their purity. The analyses were performed on Waters 1525 2998 series HPLC system (Cꢀ18
column, Sun Fire, 5 µm, 4.6 mm×150 mm) at room temperature. Methanol and water were used
as mobile phase, and the flow rate is 1.0 mL/min. The tested compounds were dissolved in
methanol, and the injection volume is 10 µL. The maximal absorbance at the range of 210ꢀ400 nm
was used as the detection wavelength.
Biological Studies
Cells and cell cultures. SMMCꢀ7721, HeLa, HepG2, HLꢀ60, and A549 cells were obtained
from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.
HLꢀ60 cells were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, and 100
units mL−1 penicillin/streptomycin and maintained in an atmosphere of 5% CO2 at 37 °C.
SMMCꢀ7721 (7721), HeLa, A549 and HepG2 cells were cultured in DMEM with 10% FBS under
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the same conditions. HEKꢀTrxR1 and HEKꢀIRES cells,72 kindly provided by Prof. Constantinos
Koumenis from University of Pennsylvania School of Medicine, were cultured in DMEM with
10% FBS, 2 mM glutamine, 100 units mL−1penicillin/streptomycin, 0.1 µM sodium selenite, and
0.4 mg/mL G418 and maintained in an atmosphere of 5% CO2 at 37 °C. The concentrations of
DMSO in all cell experiments are 0.1% (V/V). The shRNA plasmid targeting coding regions of
the TrxR1 gene (shTrxR1) and the control nontargeting shRNA (shNT)73 were kindly provided by
Prof. Constantinos Koumenis from University of Pennsylvania School of Medicine. HeLa cells
were plated in a 6ꢀwell plate with 3×105 cells/well in DMEM media without antibiotics overnight
and were transfected with either shTrxR1 (HeLaꢀshTrxR1) or shNT plasmid (HeLaꢀshNT) using
GeneTM Ⅲ efficiency Transfection reagent (Biomiga, CA, USA). After 48 h of transfection, the
cells were maintained with DMEM medium, 10% FBS, 2 mM glutamine, 100 units/mL
penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2, and selected by the
supplement with 1 ꢀg/mL puromycin for weeks.
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Cytotoxicity Assays
MTT assay.43, 44 Cells (5×103) were incubated with 13n or its derivatives in triplicate in a
96ꢀwell plate for the indicated time at 37 °C in a final volume of 100 µL. Cells treated with
DMSO alone were used as controls. At the end of the treatment (68 h), 10 µL MTT (5 mg/mL)
was added to each well and incubated for an additional 4 h at 37 °C. An extraction buffer (100 µL,
10% SDS, 5% isobutanol, 0.1% HCl) was added, and the cells were incubated overnight at 37°C.
The absorbance was measured at 570 nm on Multiskan GO (Thermo Scientific).
Trypan blue exclusion assay.43, 44 HeLa cells were seeded in 24ꢀwell plates and treated with
different concentrations of 13n for 72 h. Cells treated with DMSO alone were used as controls,
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and cell viability was determined by the trypan blue exclusion assay. After treatment, the cells
were stained with trypan blue (0.4%, w/v), and the number of viable (nonꢀstained) and dead
(stained) cells were counted under a microscope.
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Enzymes Activity Assays
GR assay.43, 44 The NADPHꢀreduced GR (0.25 U/mL) in TE buffer (50 mM TrisꢀHCl with 1
mM EDTA, pH 7.5) was incubated with different concentrations of 13n for 2 h in a 96ꢀwell plate
at room temperature in a total volume of 100 µL. Reactions were initiated by the addition of the
oxidized glutathione (GSSG) and NADPH (50 µL, final concentration: 1 mM and 400 µM,
respectively). The GR activity was determined by measuring the decrease in absorbance at 340
nm during the initial 3 min. The same amounts of DMSO were added to the control experiments
and the activity was expressed as the percentage of the control.
GPx assay.15, 74 The GPx activity was measured indirectly by a coupled reaction with GR.
GSSG, produced upon reduction of hydroperoxides by GPx, is recycled to its reduced state by GR
and NADPH. The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at
340 nm. The rate of decrease in the A340 is directly proportional to the GPx activity. To the wells
of a 96ꢀwell microliter plate were added 130 µL of TE buffer, 10 µL of a freshly prepared
NADPH solution (4.0 mM in TE buffer), 10 µL of bovine erythrocyte GPx solution (1.0
IU·mLꢀ1in TE buffer), and 10 µL of indicated concentrations of 13n. The solution was incubated
at 37 °C for 2 h. Then 10 µL of a baker’s yeast GR solution (4.0 IU·mLꢀ1in TE buffer) and 10 µL
of freshly prepared GSH solution (5.0 mM in TE buffer) were added. After adding 20 µL of H2O2
solution (5.0 mM in water), the final volume in each well was 200 µL. The background of
GPxꢀindependent NAPDH oxidation rate (r1) was determined by replacing the GPx solution with
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TE buffer. The rate of decrease in absorption of NADPH at 340 nm (r2) was measured for 4 min at
intervals of 10 sec at room temperature. The relative GPx activity was calculated by subtracting
the r1 from r2, and was expressed as the percentage of the control.
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In vitro TrxR activity by DTNB assay.43, 44 The TrxR activity was determined at room
temperature using a microplate reader. The NADPHꢀreduced TrxR (0.16 ꢀM) or U498C TrxR (3.2
µM) was incubated with different concentrations of 13n for 2 h at room temperature (the final
volume of the mixture was 50 µL) in a 96ꢀwell plate. A master mixture in TE buffer (50 µL)
containing DTNB and NADPH was added (final concentration: 2 mM and 200 µM, respectively),
and the linear increase in absorbance at 412 nm during the initial 3 min was recorded. The same
amounts of DMSO (0.1%, v/v) were added to the control experiments and the activity was
expressed as the percentage of the control.
Determination of TrxR activity in HeLa cells.43, 44 After HeLa cells were treated with
different concentrations of 13n for 48 h, the cells were harvested, and washed twice with PBS.
Total cellular proteins were extracted by RIPA buffer (50 mM TrisꢀHCl pH 7.5, 2 mM EDTA,
0.5% deoxycholate, 150 mM NaCl, 1% Triton Xꢀ100, 0.1% SDS, 1 mM Na3VO4 and 1 mM
PMSF) for 30 min on ice. The total protein content was quantified using the Bradford procedure.
TrxR activity in cell lysates was measured by the endpoint insulin reduction assay. Briefly, the cell
extract containing 20 µg of total proteins was incubated in a final reaction volume of 50 µl
containing 100 mM TrisꢀHCl (pH 7.6), 0.3 mM insulin, 660 µM NADPH, 3 mM EDTA, and 15
µM E. coli Trx for 30 min at 37 °C. The reaction was terminated by adding 200 µL of 1 mM
DTNB in 6 M guanidine hydrochloride, pH 8.0. A blank sample, containing everything except Trx,
was treated in the same manner. The absorbance at 412 nm was measured, and the blank value
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was subtracted from the corresponding absorbance value of the sample. The same amounts of
DMSO were added to the control experiments and the activity was expressed as the percentage of
the control.
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Imaging TrxR activity in HeLa cells by TRFS-green.15, 41 HeLa cells were treated with
indicated concentrations of 13n for 44 h, followed by treatment with TRFSꢀgreen (10 µM) for
additional 4 h. The intensity of the green fluorescence in the cells correlates with the cellular TrxR
activity. Phase contrast (top panel) and fluorescence (bottom panel) images were acquired with
FloidTM cell imaging station (Life Technology).
Assessment of the intracellular ROS.43, 44 HeLa cells were plated in 12ꢀwell plates and were
treated with 13n for 1 h. After removal of the medium, the ROS indicator DCFHꢀDA (10 µM) in
fresh FBSꢀfree medium was added, and continued incubation for 30 min at 37 °C in dark. The
cells were visualized and photographed under a Leica inverted fluorescent microscopy.
Apoptosis Assays
Hoechst 33342 Staining.43, 44 HeLa cells were plated in 12ꢀwell plates and were incubated
with different concentrations of 13n for 12 h followed by addition of Hoechst 33342 to a final
concentration of 5 µg/mL. After incubation for additional 20 min, the cells were visualized and
photographed under a Leica inverted fluorescent microscopy.
Measurement of caspase 3 activity.43, 44 HeLa cells were treated with different concentrations
of 13n for 24 h in 100ꢀmm dishes. The cells were collected, washed twice with PBS, and then
lysed with RIPA buffer for 30 min on ice. The protein content was quantified using the Bradford
procedure. A cell extract containing 50 µg of total proteins was incubated with the assay mixture
(50 mM Hepes, 2 mM EDTA, 5% glycerol, 10 mM DTT, 0.1% CHAPS, 0.2 mM AcꢀDEVDꢀpNA,
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pH 7.5) for 3 h at 37 °C in a final volume of 100 µL. The absorbance at 405 nm was measured
using a microplate reader. The same amounts of DMSO were added to the control experiments
and the activity was expressed as the percentage of the control.
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Annexin V/PI staining.43, 44 HeLa cells were treated with varying concentrations of 13n for
24 h in 60ꢀmm dishes. The cells were harvested and washed with PBS. Apoptotic cells were
identified by double staining with FITCꢀconjugated Annexin V and PI according to the
manufacturer’s instructions (Zoman Biotech, Beijing, China). The cells show four different cell
populations marked as follows: doubleꢀnegative (unstained) cells showing live cell population,
FITCꢀpositive and PIꢀnegative stained cells showing early apoptosis, FITC/PI doubleꢀstained cells
showing late apoptosis, and finally PIꢀpositive and FITCꢀnegative stained cells showing necrotic
cells. Both the early apoptotic cells and the late apoptotic cells were designated as apoptotic cells.
Data were obtained and analyzed using Cellometer K2 Image Cytometer (Nexcelom Biosciences).
Statistics
Data are presented as mean ± S. E. Statistical differences between two groups were assessed
by the Students t-test. Comparisons among multiple groups were performed using oneꢀway
analysis of variance (ANOVA), followed by a post hoc Scheffe test. P<0.05 was used as the
criterion for statistical significance.
ASSOCIATED CONTENT
Supporting Information
Inhibition of TrxR in vitro by different compounds, detection of cellular thiols after 13n treatment,
determination of total glutathione after BSO treatment, and detailed synthetic procedures and
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characterization of the compounds. This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: fangjg@lzu.edu.cn; Tel: +86 931ꢀ8912500.
ACKNOWLEDGMENTS
The financial supports from Lanzhou University (the Fundamental Research Funds for the
Central Universities, lzujbkyꢀ2014ꢀ56), and Natural Science Foundation of Gansu Province
(145RJZA225) are greatly acknowledged. The authors also express appreciation to Professor Arne
Holmgren from Karolinska Institute for the recombinant rat TrxR1, and Professor Constantinos
Koumenis from the University of Pennsylvania School of Medicine for shRNA plasmids,
HEKꢀTrxR1 and HEKꢀIRES cells.
ABBREVIATIONS USED
BSO: buthionine sulphoximine; DCFH: dichlorodihydrofluorescin; DCFHꢀDA: 2’,
7’ꢀdichlorofluorescin diacetate; DTT: DLꢀdithiothreitol; EWG: electronꢀwithdrawing group; FITC:
fluoresceinꢀ5ꢀisothiocyanate; GPx: glutathione peroxidase; GR: glutathione reductase; GSH:
reduced glutathione; GSSG: oxidized glutathione; HEKꢀIRES: HEK cells transfected with a
vector; HEKꢀTrxR1: HEK cells stably overexpressing TrxR1; HeLaꢀshNT: HeLa cells stably
transfected with a nonꢀtargeting shRNA; HeLaꢀshTrxR1: HeLa cells stably knocking down TrxR1;
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MTT:
3ꢀ(4,5ꢀdimethylꢀ2ꢀthiazolyl)ꢀ2,5ꢀdiphenylꢀ2ꢀHꢀtetrazolium
bromide;
NAC:
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Nꢀacetylcysteine; PI: propidium iodide; ROS: reactive oxygen species; Sec: selenocysteine; TE
buffer: 50 mM TrisꢀHCl with 1 mM EDTA, pH 7.5; Trx: thioredoxin; TrxR: thioredoxin reductase;
Xn: xanthohumol.
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