Sanguinarine as a new chemical entity of thioredoxin reductase inhibitor to elicit oxidative stress and promote tumor cell apoptosis

Article abstract of DOI:10.1016/j.freeradbiomed.2020.01.008

The alteration of redox homeostasis is a hallmark of cancer cells. As a critical player in regulating cellular redox signaling, thioredoxin reductase (TrxR) enzymes are increasingly recognized as attractive targets for anticancer drug development. We repo

Full text of DOI:10.1016/j.freeradbiomed.2020.01.008

Journal Pre-proof
Sanguinarine as a new chemical entity of thioredoxin reductase inhibitor to elicit
oxidative stress and promote tumor cell apoptosis
Juan Yao, Dongzhu Duan, Zi-Long Song, Junmin Zhang, Jianguo Fang
PII:
S0891-5849(19)32389-5
DOI:
https://doi.org/10.1016/j.freeradbiomed.2020.01.008
FRB 14561
Reference:
To appear in: Free Radical Biology and Medicine
Received Date: 25 November 2019
Revised Date: 8 January 2020
Accepted Date: 8 January 2020
Please cite this article as: J. Yao, D. Duan, Z.-L. Song, J. Zhang, J. Fang, Sanguinarine as
a new chemical entity of thioredoxin reductase inhibitor to elicit oxidative stress and promote
tumor cell apoptosis, Free Radical Biology and Medicine (2020), doi: https://doi.org/10.1016/
j.freeradbiomed.2020.01.008.
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Sanguinarine as a new chemical entity of thioredoxin reductase inhibitor to elicit oxidative
stress and promote tumor cell apoptosis
a, b
a,
c
a
a
a*
Juan Yao, Dongzhu Duan , Zi-Long Song, Junmin Zhang, and Jianguo Fang
a
State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical
Engineering, Lanzhou University, Lanzhou, Gansu 730000, China.
b
School of pharmacy, Gansu University of Chinese Medicine, Lanzhou, Gansu 730000, China.
c
Shaanxi Key Laboratory of Phytochemistry, Baoji University of Arts and Sciences, Baoji, 721013,
China.
*Corresponding author, E-mail: fangjg@lzu.edu.cn (J. Fang); Fax: +86 931 8915557.
1

ABSTRACT
The alteration of redox homeostasis is a hallmark of cancer cells. As a critical player in
regulating cellular redox signaling, thioredoxin reductase (TrxR) enzymes are increasingly
recognized as attractive targets for anticancer drug development. We reported herein the natural
product sanguinarine (SAN) as a potent inhibitor of TrxR with a new chemical scaffold. Inhibition of
TrxR leads to accumulation of the oxidized thioredoxin, elicits oxidative stress, and finally promotes
apoptosis of cancer cells. Further synthesis of different model compounds of SAN demonstrated that
the phenanthridinium unit is responsible for the TrxR inhibition. The core structure of SAN, e.g., the
phenanthridinium moiety, is different from those of known TrxR inhibitors, and thus SAN is a new
chemical entity of TrxR inhibitors and may serve a lead for further development. In addition, as the
phenanthridinium scaffold is widely present in natural products, the disclosure of TrxR inhibition by
such unit sheds light in understanding the pharmacological actions of these molecules.
Table of Contents graphic
Keywords: Thioredoxin; Sanguinarine; Phenanthridinium; Oxidative stress; Apoptosis
2

Introduction
Natural products and their synthetic analogues are a major source of therapeutic agents, and
have been used for centuries to treat a variety of human ailments[1, 2]. With the development of
modern molecular medicine, an increasing number of active components from herbal plants have
been identified and their putative cellular targets have been disclosed. Sanguinarine (SAN) is mainly
present in Sanguinaria canadensis, but also found in Chelidonium majus and Macleaya cordata. As a
major bio-active component that belongs to the benzophenanthridine alkaloid family of these herb
plants, SAN has been demonstrated to exhibit a wide array of pharmacological activities, including
anti-hypertensive [3], anti- osteoporosis [4] and anti-microbial [5] activities. Historically, plants
containing SAN were believed to have anticancer properties. Although SAN was isolated in the late
1940s [6], extensive studies on the mechanisms underlying SAN-mediated tumor suppression begun
only recently. SAN displayed significant cytotoxicity on different types of cancer cells with various
action mechanisms [7, 8], such as upregulation of miR-16-2 [9], inhibition of the JaK2/STAT3
signaling [10], activation of ERK1/2 [11], dissociation of STRAP-MELK complex [12], suppression
of sonic hedgehog-Gli-Nanog pathway [13], and binding to nucleic acids [14, 15]. It is worth of
noting that most of the cellular actions of SAN involved the induction of ROS production and
oxidative stress in cancer cells [9, 11-13, 16]. However, the molecular mechanisms of ROS induction
by SAN are not clear.
Mammalian thioredoxin reductase (TrxR) isoenzymes are selenoproteins that function to
maintain thioredoxin (Trx) proteins in a reduced state via supplying electrons from NADPH [17, 18].
The C-terminal selenocysteine (Sec) is a critical residue for the functions of TrxR. TrxR is a major
antioxidant enzyme, and its inhibition usually leads to elevation of cellular ROS and induction of
oxidative stress [19, 20]. Besides the critical role of TrxR in redox regulation, mounting evidence has
supported that TrxR is essentially involved in multiple steps of tumorigenesis and is a promising
target for anticancer drug development [17, 21-30]. We thus hypothesized that SAN may interact
with TrxR to induce oxidative stress and kill cancer cells. As our continuing efforts in discovering
small therapeutic molecules that interfere cellular redox signaling [31-44], we reported herein that
SAN is a potent inhibitor of TrxR with the ability to inhibit both the recombinant enzyme and the
enzyme in HeLa cells. Inhibition of TrxR by SAN promotes upregulation of ROS level and
accumulation of oxidized Trx, and eventually elicits apoptosis of HeLa cells. Silence of TrxR
3

expression by RNA interference enhances the cytotoxicity of SAN while overexpression of the
enzyme attenuates the cytotoxicity of SAN, supporting that TrxR is essentially involved in the
cellular actions of SAN. The discovery of targeting TrxR by SAN provides insights for the
pharmacological activity of SAN, and accounts for the ROS induction ability by SAN in cells. The
core structure of SAN, i.e., the phenanthridinium scaffold (Figure 1A), is widely present in many
natural products, and the disclosure of the inhibition of TrxR by SAN may also shed lights in
understanding the action mechanisms of the related molecules. More importantly, the active motif of
SAN is different from the known TrxR inhibitors, thus SAN could serve a lead for development of
novel TrxR inhibitors.
Results and Discussion
Inhibition of TrxR by SAN.
Incubation of the reduced rat recombinant TrxR1 with SAN causes a dose-dependent inhibition
of the enzyme activity determined by using 5, 5’-dithiobis-2-nitrobenzoic acid (DTNB) as an
artificial substrate (Figure 1B). SAN showed weak inhibition to GR, a homologue of TrxR, and little
th
effect on U498C TrxR1, a mutant TrxR where the 498 Sec was replaced by Cys. These results
indicated that the Sec residue in TrxR is involved for the inhibition of TrxR by SAN. The inhibition
of TrxR by SAN was further confirmed by the Trx-mediated insulin reduction assay (Figure 1C), and
a consistent inhibition of the enzyme activity was observed. Next, we employed two di� erent assays,
i.e., the classic Trx-mediated insulin reduction assay and our recently developed and image-based
live cell TrxR activity assay [45, 46], to determine the inhibition of TrxR in a cellular content. As
shown in Figure 1D and Figure 1E, both assays gave consistent results showing a dose-dependent
inhibition of TrxR by SAN in HeLa cells. No apparent change of TrxR1 protein expression was
observed after the cells were treated with SAN for 12 h (Figure S1B, Supporting Information),
indicating that the decreased TrxR enzyme activity after SAN treatment is due to the direct inhibition
of the enzyme. Taken together, these data clearly demonstrated that SAN is a potent TrxR inhibitor.
We observed a dimer band (~110 kD) that is independent of SAN-treatment appeared in samples
(Figure S1B). As we run the samples under reducing conditions, it is not like this band is formed via
intermolecular disulfide bond. As reported by Arner et al. [47], this band is likely formed via the
oxidation of the Trp114 residue in TrxR. Also Arner and colleagues observed formation of
4

TrxR1-Trx1 complex in the cisplatin-treated cells [48]. However, the band corresponding to the
crosslink of TrxR1 and Trx1 (~70 kD) was not observed (Figure S1B).
Accumulation of ROS and oxidized Trx in cells
The major function of TrxR is to maintain Trx in a reduced state and defend against oxidative
stress. Having confirmed that SAN is a potent TrxR inhibitor, we next determined the ROS level and
Trx redox states in HeLa cells. A general ROS probe 2’,7’-dichlorofluorescin diacetate (DCFH-DA)
and a superoxide-specific probe dihydroethidium (DHE) were loaded to stain cells. As shown in
Figure 2A and Figure 2B, a gradual elevation of ROS level in cells treated with increasing
concentrations of SAN was observed, indicating that SAN had the ability to promote ROS
accumulation in cells. Trx is a small redox protein that may directly scavenge ROS or indirectly
serve a reductase by supplying reducing equivalents to other antioxidant proteins, such as
peroxiredoxins and methionine sulfoxide reductases. We then determined the redox status of Trx by
phenylarsenoxide (PAO) pulldown assay developed in our group (Figure 2C). The
tris(2-carboxyethyl)phosphine (TCEP)- and diamide-treated samples, corresponding to the fully
reduced and oxidized Trx proteins, were prepared as positive controls. Trx1 is predominantly present
as a reduced state in the vehicle-treated cells. However, after SAN treatment, the oxidized Trx1
increased remarkably. Quantification of the ratio of reduced Trx1 to oxidized Trx1 was shown in
Figure 2D. After the SAN treatment, total Trx1 proteins decreased slightly (Figure S1A, Supporting
Information). The redox states of Trx play a critical role in regulating apoptosis, and reduced Trx
may bind to several target proteins to inhibit apoptosis while oxidized Trx losses such
apoptosis-inhibiting function. Thus, the accumulation of oxidized Trx contributes to the induction of
apoptosis by SAN (vide infra).
Induction of apoptosis by SAN
SAN displays significant cytotoxicity to HeLa cells in dose- and time-dependent manners
(Figure 3A). The mechanism of such cytotoxicity was due to the induction of apoptosis, which was
supported by three different assays (Figures 3B-D). First, the Annexin V-FITC/PI double staining
assay showed that a significant amount of apoptotic cells was detected after SAN treatment (Figure
3B). Second, the activation of the caspase-3 was determined, and it was found that SAN activated the
enzyme activity dose-dependently (Figure 3C). Third, the mitochondrial membrane potential (MMP)
was evaluated by a fluorescent probe Rhodamine 123, and a gradual decrease of the fluorescence,
5

indicating a collapse of MMP, was observed upon SAN treatment (Figure 3D).
Contribution of Targeting TrxR to cytotoxicity of SAN
As we have fully demonstrated that SAN inhibited both the pure TrxR enzyme and the enzyme
in cells (Figure 1), we next asked the physiological significance of TrxR inhibition to the cellular
actions of SAN. We generated two stable cell lines, i.e., HeLa-shNT cells and HeLa-shTrxR1 cells,
by transfecting a non-targeting shRNA plasmid and a plasmid specifically targeting TrxR1 into HeLa
cells [44]. The knockdown efficiency was validated in our previous publications [38, 44]. As shown
in Figure 3E, SAN had significantly higher toxicity to HeLa-shTrxR1 cells than HeLa-shNT cells at
concentrations of 0.5 and 1.0 µM. The inset in Figure 3E showed the knockdown efficiency of TrxR1
protein expression. In contrast to the loss-of-function assay, overexpression of the functional TrxR1
protein in HEK cells lowered the cytotoxicity of SAN (Figure 3F). The inset showed the expression
of TrxR1 protein in HEK cells. The activity of TrxR in the cells was further confirmed by both the
classic Trx-mediated insulin reduction assay and the image-based assay using the specific TrxR
probe TRFS-green (Figure S2, Supporting Information). Taken together, our data supported that the
cytotoxicity of SAN is related to its interaction with TrxR.
Phenanthridinium moiety as a new scaffold for TrxR inhibition
As TrxR is involved in multiple steps of tumorigenesis, targeting its inhibition is promising in
treatment of cancer [17, 21, 22]. We have thoroughly reviewed the published and patented TrxR
inhibitors, and most of TrxR inhibitors, such as Michael acceptors, metal complexes and disulfides,
harbor an electrophilic center [49-51]. The essential redox active Sec residue at the C-terminus of
TrxR is highly nucleophilic, and may react with electrophilic centers of these inhibitors, leading to
inhibition of TrxR. The structure of SAN is different from all the known TrxR inhibitors. We
speculated that the phenanthridinium moiety could be an active motif for the inhibition of TrxR as
the α-carbon next to the quaternary nitrogen may be considered as an electrophilic center (Figure
4A), and this electrophilicity has been involved in the inhibition of the aminotransferase activity [52]
and formation of DNA adducts [53]. To further understand the inhibition mechanism of SAN, we
prepared three model compounds 5, 6 and 7 (Scheme 1), and determined their ability to inhibit TrxR.
The three compounds were obtained with moderate yields. Compound 5 was prepared in four steps
using 1-naphthylamine as a starting material. Methylation of compound 5 affords compound 6,
which was further reduced with sodium borohydride (NaBH ) yielding compound 7. As shown in Fig.
4
6

4B, compound 6 showed dose-dependent inhibition of TrxR, while compounds 5 and 7 had little
effect. These data supported that the phenanthridinium scaffold is responsible for the inhibition of
TrxR by SAN.
One of the hallmarks of mammalian TrxR enzymes is that they uniformly harbor a critical Sec
residue in their C-terminal active sites. Compared to its homologue Cys, Sec has stronger
nucleophilicity and is more resistant to irreversible oxidation [54-56]. The high nucleophilicity of the
Sec residue renders the enzyme vulnerable to be modified by electrophiles, based on which
numerous selective TrxR inhibitors have been developed [17, 49, 51]. The observed strong inhibition
of TrxR but not U498C TrxR, and weak inhibition of GR (Fig. 1B) could also attribute to this
character of Sec. We believed that installation of different substitute groups on the
benzophenanthridine ring to tune the electrophilicity of the iminium bond and weak interactions with
TrxR may further improve the selective inhibition of TrxR. Thus, SAN serves a structurally-novel
lead to advance development of TrxR inhibitors.
The anticancer activity of SAN has been extensively studied, and the induction of oxidative
stress is demonstrated to be involved in the underlying mechanisms of its cellular actions [9, 11-13,
16]. However, how the oxidative stress is elicited is not clear. We reported here that SAN efficiently
inhibits TrxR, induces accumulation of ROS and oxidized Trx, and eventually leads to oxidative
stress-mediated apoptosis. As TrxR is a critical player of cellular redox regulation, its inhibition may
account for the elicitation of oxidative stress by SAN. In addition, as the core structure, i.e., the
phenanthridinium scaffold, is widely distributed in many natural products, the clarification of TrxR
inhibition by SAN may further shed lights in understanding the cellular actions of these molecules.
More importantly, the phenanthridinium moiety was disclosed for the first time as an active motif for
potent TrxR inhibition, thus opening a new window in designing and constructing novel TrxR
inhibitors.
Materials and Methods
Chemicals and enzymes
SAN (purity >98%) was obtained from Baoji Herbest Bio-Tech Co., Ltd (Baoji, China). A stock
solution (100 mM) was prepared in dimethyl sulfoxide (DMSO) and kept at -20 °C. The recombinant
7

U498C TrxR1 mutant (Sec→Cys) was essentially prepared as described [42, 57]. Recombinant rat
TrxR1 is a gift from Prof. Arne Holmgren at Karolinska Institute, Sweden. E. coli Trx was purchased
from IMCO (Stockholm, Sweden, www.imcocorp.se). NADPH was obtained from Roche
(Mannheim, Germany). Dulbecco's modified Eagle’s medium (DMEM), bovine insulin,
N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA), G418, yeast glutathione reductase (GR),
oxidized glutathione (GSSG), 2,3-dimercapto-1-propanesulfonic acid (DMPS), TCEP, Hoechst
3
3342, Rhodamin123, dimethyl sulfoxide (DMSO), DTNB, DHE and DCFH-DA were purchased
from Sigma-Aldrich (St. Louis, USA). Fetal bovine serum (FBS) was obtained from HyClone
Logan, USA). Primary antibodies against TrxR1 (anti-TrxR1) and Trx1 (anti-Trx1) were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 3-(4, 5-dimethylthiazol-2-yl)-2,
-diphenyltetrazolium bromide (MTT), penicillin and streptomycin were obtained from Amresco
Solon, USA). Phenylmethylsulfonyl fluoride (PMSF), bovine serum albumin (BSA), sodium
orthovanadate(V) (Na VO ) and anti-actin antibody were purchased from Beyotime (Nantong,
(
5
(
3
4
China). HEK cells stably overexpressing TrxR1 (HEK-TrxR1), HEK cells transfected with a vector
HEK-IRES) and shRNA plasmids (non-targeting control, shNT; shRNA plasmids targeting TrxR1,
(
shTrxR1) are gifts from Prof. Constantinos Koumenis (University of Pennsylvania School of
Medicine) [58]. The preparation of HeLa-shTrxR1 cells and HeLa-shNT cells were described
elsewhere.[44]
Chemistry
The synthesis of model compounds was illustrated in Scheme 1, and the original NMR and spectra of
the synthesized compounds were included in the Supporting Information (Figures S3-S16).
Synthesis of compound 1. 1-Naphthylamine (1 eq) and dry pyridine (2 eq) was dissolved in
anhydrous THF, the mixture of which was then under ice bath followed by addition of TsCl (1.1 eq).
The reaction was stirred under room temperature overnight. The reaction was monitored by TLC,
and was quenched by saturated NaHCO aqueous solution when the reaction is complete. The
3
mixture was extracted by ethyl acetate for three times, and the combined organic phase was dried by
Na SO and concentrated under reduced pressure. The crude residue was used for next step without
2
4
future purification.
Synthesis of compound 3. Compound 1 (2 eq), compound 2 (2.2 eq), K CO (3 eq), and NaI (0.1 eq)
2
3
8

were added into a flask containing acetone, the mixture of which was then heated to reflux for 6
hours. The reaction was allowed to cool and was extracted using ethyl acetate. The organics were
dried over Na SO , filtered and the volatiles were evaporated under reduced pressure. The residues
2
4
were then purified via silica gel column chromatography to afford the target compound with a yield
1
of 81.5%. H NMR (CDCl , 400 MHz) δ: 7.987 (d, 1H, J=8.0 Hz), 7.749-7.730 (m, 2H), 7.627 (d,
3
2
1
H, J=8.0 Hz), 7.520-7.490 (m, 1H), 7.415-7.337 (m, 2H), 7.289-7.253 (m, 4H), 7.121-7.084 (m,
H), 6.984-6.919 (m, 2H), 5.204 (d, 1H, J=14 Hz), 4.829 (d, 1H, J=14 Hz), 2.449 (s, 3H); MS-ESI
+
m/z: 466.1 [M+H] .
Synthesis of compound 4 [59]. Pulverescent K CO (2 eq.), compound 3 (1 equiv.), Pd(OAc) (0.03
2
3
2
eq) and PCy –HBF (0.06 eq) were placed in a schlenk tube equipped with a magnetic stir. The tube
3
4
was purged with argon and the degassed N, N-dimethylacetamide (DMA) was added. The reaction
was heated to 130 ºC for 16 hours. Then the reaction mixture was allowed to cool to room
temperature. The crude mixture was then evaporated directly under reduced pressure to dryness,
which was loaded onto silica and purified by chromatography to afford the target compound with a
1
yield of 89.7%. H NMR (DMSO-d , 400 MHz) δ: 8.507 (d, 1H, J=8.4 Hz), 7.996-7.957 (m, 2H),
6
7.849 (d, 1H, J= 8.8 Hz), 7.644-7.555 (m, 2H), 7.401 (d, 1H, J=7.2 Hz), 7.219-7.092 (m, 3H), 6.788
13
(
s, 4H), 5.232 (d, 1H, J=16.8 Hz), 4.611 (d, 1H, J=14 Hz), 2.139 (s, 3H); C NMR (DMSO-d , 100
6
MHz) δ: 143.063, 133.422, 133.332, 131.986, 131.679, 131.017, 130.909, 128.999, 128.464,
1
1
28.438, 128.063, 127.546, 127.386, 127.080, 126.468, 126.321, 126.251, 126.178, 123.245,
+
21.903, 50.160, 20.833; MS-ESI m/z: 386.0 [M+H] .
Synthesis of compound 5 [60]. To a stirred solution of freshly prepared Na/naphthalene (5 eq) in
anhydrous THF was added compound 5 (1 eq) in anhydrous THF at −78 °C and the resulting mixture
was stirred at the same temperature for 2 h. Then oxygen was bubbled into the reaction mixture,
which was held at room temperature for 24 hours. The reaction was quenched with saturated aqueous
solution of NH Cl and then extracted with ethyl acetate. The combined organic layers were dried
4
with Na SO , filtered, and concentrated under reduced pressure. The crude residue was purification
2
4
1
by column chromatography on silica gel
to afford the target compound with a yield of 66.3%. H
NMR (DMSO-d , 400 MHz) δ: 9.569 (s, 1H), 9.315 (d, 1H, J=8.0 Hz), 8.856 (d, 1H, J=8.0 Hz),
6
8.739 (d, 1H, J=8.8 Hz), 8.290 (d, 1H, J=8.0 Hz), 8.135-8.076 (m, 2H), 7.974-7.933 (m, 1H),
1
3
7
.820-7.703 (m, 3H); C NMR (DMSO-d , 100 MHz) δ: 152.288, 140.561, 132.910, 132.092,
6
9

1
1
31.336, 128.759, 127.822, 127.741, 127.652, 127.480, 127.008, 126.521, 124.227, 122.575,
+
20.673, 120.549; MS-ESI m/z: 230.0 [M+H] .
Synthesis of compound 6. Compound 5 (1 eq) was dissolved in anhydrous toluene in a schlenk tube
equipped with a magnetic stir, and CF SO CH (20 eq) was added to the reaction. Then the reaction
3
3
3
tube was sealed, which was heated to 120 °C for 4 hours. Then the reaction mixture was allowed to
cool to room temperature, and the precipitate was filtered, washed with toluene and diethyl ether, and
1
then collected as pure product to afford the target compound with a yield of 74.8%. H NMR
(
DMSO-d , 400 MHz) δ: 9.697 (s, 1H), 8.734-8.658 (m, 2H), 8.475 (d, 1H, J=8.0 Hz), 8.372 (d, 1H,
6
1
3
J=8.0 Hz), 8.254 (t, 1H, J=8.0 Hz), 8.096-7.990 (m, 3H), 7.838-7.802 (m, 2H), 4.863 (s, 3H); C
NMR (DMSO-d , 100 MHz) δ: 155.532, 137.648, 134.636, 133.068, 131.814, 131.526, 130.718,
6
1
2
29.353, 129.131, 127.739, 126.881, 126.124, 123.900, 123.540, 120.160, 52.588; MS-ESI m/z:
+
45.0 [M+H] .
Synthesis of compound 7. Compound 6 (1eq) was suspended in MeOH, which was cooled to 0 °C
followed by addition of NaBH (3 eq). 15 minutes later, MeOH was removed and the residue was
4
directly loaded onto silica gel and purified by chromatography to afford the target compound with a
1
yield of 78.2%. H NMR (CDCl , 400 MHz) δ: 8.400 (d, 1H, J=8.0 Hz), 7.955 (d, 1H, J=8.0 Hz),
3
7
7
1
1
.883-7.853 (m, 2H), 7.716 (d, 1H, J=8.8 Hz), 7.599-7.558 (m, 1H), 7.531-7.491 (m, 1H),
13
.462-7.315 (m, 3H), 4.287 (s, 2H), 2.729 (s, 3H); C NMR (CDCl3, 100 MHz) δ: 144.310, 134.338,
32.307, 132.105, 129.447, 128.348, 127.808, 127.663, 127.016, 126.233, 126.128, 125.440,
+
24.796, 124.249, 122.942, 121.954, 55.216, 41.582; MS-ESI m/z: 246.3 [M+H] .
Biology
Cell cultures. HeLa cells were obtained from the Shanghai Institute of Biochemistry and Cell
Biology, Chinese Academy of Sciences, and were grown in DMEM supplemented with 10 % FBS, 2
−
1
mM glutamine, 100 units·mL penicillin/streptomycin in a 37 °C incubator with a humidified, 5%
CO atmosphere (standard culture conditions). HEK-TrxR1 and HEK-IRES cells were cultured under
2
the standard culture conditions supplemented with additional 0.1 µM sodium selenite and 0.4
−
1
mg·mL G418. HeLa-shTrxR1 and HeLa-shNT cells were generated as described [44] and
-
1
maintained under the standard culture conditions supplemented with additional 1 µg·mL
puromycin.
1
0

3
MTT assay. The cytotoxicity of SAN in vitro was measured using the MTT assay. Briefly, 5×10
cells were incubated with SAN or other agents in triplicate in a 96-well plate for different times at
7 °C in a final volume of 100 µL. We used the DMSO-treated cells as the control group. At the end
3
−
1
of the treatment, 10 µL MTT (5 mg·mL ) was added to each well and incubated for 4 h at 37 °C,
then added 100 µL extraction buffer (10% SDS, 5% iso-butanol, 0.1% HCl) each well, and the cells
were incubated overnight at 37°C. The absorbance was measured at 570 nm using a microplate
reader (Thermo Scientific Multiskan GO, Finland).
In vitro TrxR activity assays [32, 44]. The TrxR activity was determined by DTNB assay and
endpoint insulin reduction assay at room temperature using a microplate reader. For DTNB assay,
the NADPH-reduced TrxR (85 nM) or U498C TrxR (350 nM) was incubated with different
concentrations of SAN for 2 h at room temperature a final volume of 50 µL (the final volume of the
mixture was 50 µL). Then added 50 µL TE buffer (50 mM Tris-HCl, 1 mM EDTA, pH 7.5)
containing 200 µM NADPH and 2 mM DTNB, recorded the linear increase during the initial 3 min
in absorbance at 412 nm. For endpoint insulin reduction assay, the NADPH-reduced TrxR (170 nM)
was incubated with different concentrations of SAN in 96-well plate (the final volume of the mixture
was 50 µL) at room temperature for 2 h. A master mixture (50 µL TE buffer containing 4 µM E. coli
Trx, 0.32 mM insulin and 0.4 mM NADPH) was added to the solution, and the incubation continued
at room temperature for 30 min. Addition of 100 µL 1 mM DTNB in 6 M guanidine hydrochloride
(pH 8.0) terminated the reaction and the absorbance at 412 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.
In vitro GR assay. The NADPH-reduced GR (0.25 U) in TE buffer was incubated with different
concentrations of SAN for 2 h in a 96-well plate (the total volume was 50 µL) at room temperature.
Reactions were initiated by the addition of 50 µL TE buffer containing 400 µM NADPH and 1 mM
GSSG. The GR activity was assayed by measuring the decrease in absorbance at 340 nm during the
initial 3 min.
Determination of redox states of Trx [39]. After HeLa cells (70-80% confluence) were treated with
SAN for 12 h, the cells were harvested and washed twice with PBS. Treat HeLa cells include control
group, fully oxidized group, fully reduced group and drug group. Total cellular proteins were
extracted with RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% TritonX-100,
1
1

0
.5% deoxycholate, 0.1% SDS, 1 mM PMSF and 1 mM Na VO ) for 30 min on ice. Two control
3 4
groups, i.e., the fully oxidized or reduced groups, were set for this assay by treatment of the
non-treated cell lysate with diamide (5 mM) or TCEP (5 mM) for 30 min, respectively. The total
protein content was quantified using the Bradford procedure. All samples were incubated with
Sepharose-PAO (Sepharose-PAO was prepared in our group [42]) at 37 °C for 30 min with vortexing
occasionally. The supernatant (containing oxidized Trx) was transferred to a new tube. The beads
were (containing reduced Trx) washed three times with TE, and the bound protein were knocked out
by DMPS (20 mM). All samples were subjected SDS-PAGE, followed by western blotting detection
of Trx proteins.
Determination of TrxR activity in HeLa cells
Trx-mediated endpoint insulin reduction assay. HeLa cells were treated with different concentrations
of SAN for 12 h. The cells were harvested, and total cellular proteins were extracted by RIPA buffer.
The protein concentration was quantified by the Bradford procedure. TrxR activity in cell lysates
was examined by the endpoint insulin reduction assay described as previous [32, 44].
Live cell imaging by the specific TrxR probe TRFS-green [45, 46]. HeLa cells were plated in 12-well
plates and were treated with 0, 1, 2, 3, and 5 µM SAN for 8 h. Then added 10 µM TRFS-green and
incubated the cells for another 4 h. The cells were rinsed with the medium to remove the remaining
TRFS-green. The fluorescence images were acquired by a fluorescent microscope (Floid Cell
Imaging Station).
Western Blotting analysis. HeLa cells were plated in 100-mm culture dishes and treated with various
concentrations of SAN. After appropriate time, the cells were harvested and total cellular proteins
were extracted by RIPA buffer. Equal amounts of protein in each sample were separated by
SDS-PAGE followed by western blotting analysis. After blocking with 5 % non-fat milk at room
temperature for 1 h, then PVDF membranes were incubated at 4 °C overnight with antibodies against
TrxR1 (1:1000), Trx1 (1:1000) and actin (1:1000) primary antibodies. After washing membranes
with TBST for three times, and then incubated membranes with the secondary antibodies (1:2000)
for 1 h at room temperature. The signal was visualized by ECL chemiluminescence (GE Healthcare,
Buckinghamshire, UK).
Intracellular ROS measurement. ROS level was assessed using the fluorescent dye DCFH-DA or
DHE. HeLa cells were seeded in a 12-well plate and allowed to adhere. After HeLa cells were
1
2

treated with 0, 1, 2 or 3 µM SAN for 6 h, removed the medium and subsequently incubated with 10
µM DCFH-DA or 10 µM DHE in fresh FBS-free medium for 30 min at 37 °C in dark. Images were
then visualized and captured by fluorescent microscope (Floid Cell Imaging Station).
Apoptosis assays
Annexin V-FITC/PI staining. HeLa cells were seeded in 6-well plates and treated with different
concentrations of SAN for 12 h. The cells were harvested and washed with PBS. According to the
Annexin V-FITC/PI double staining apoptosis detection kit (Zoman Biotech, Beijing, China),
apoptotic cells was determined and analyzed by flow cytometry (BD Biosciences, USA) with Cell
Quest software (BD Biosciences).
Caspase-3 activity assay. As described by us previously [43], we applied N-acetyl-Asp-Glu-Val-
Asp-p-nitroanilide (Ac-DEVD-pNA) as a substrate to measure the activity of caspase-3. Briefly,
HeLa cells were treated with different concentrations of SAN for 12 h. Then the cells were collected
and lysed with RIPA buffer, using the Bradford procedure to quantify the protein content. Then, 15
µg of total proteins were incubated with the assay mixture (50 mM Hepes, 0.2 mM Ac-DEVD-pNA,
2
1
mM EDTA, 10 mM DTT, 5% glycerol, 0.1% CHAPS, pH 7.5) at 37°C for 2 h in a final volume of
00 µL. The absorbance at 405 nm was read by a microplate reader.
Measurement of MMP [61]. Rhodamine123 is a cationic fluorescent probe that has been used for the
measurement of MMP. HeLa cells were seeded in 6-well plates and allowed to adhere for 24 h. The
cells were incubated with the indicated concentrations of SAN for an additional 12 h followed by
addition of 5 µM Rhodamine123 for 20 min at 37°C in an incubator and then analyzed by means of
flow cytometry (Becton Dickinson, San Jose, CA).
Statistics
All experiment data are presented as the mean ± SE. Statistical differences between two groups
were assessed by Student’s t test. P<0.05 was used as the criterion for statistical significance.
Conclusions
We have elucidated TrxR as a target of SAN both in vitro and in human hepatocellular
carcinoma HeLa cells, and demonstrated that SAN promotes oxidative stress-mediated apoptotic cell
death through an unprecedented mechanism. Since SAN belongs to the large family of
1
3

phenanthridinium natural products, the disclosure of its target of TrxR inhibition sheds light in
understanding the pharmacological functions of phenanthridinium natural products. Furthermore, our
results revealed for the first time that the core structure of SAN, e.g., the phenanthridinium moiety,
could serve a new scaffold to develop novel TrxR inhibitors.
Associated Content
Supporting Information
Supplementary figures, original NMR and MS spectra. This material is available free of charge via
the Internet www.xxx.
Acknowledgments
The financial supports from the National Natural Science Foundation of China (21572093 &
21778028), the 111 project and the Natural Science Foundation of Gansu Province (18JR4RA003)
are greatly acknowledged. The authors also express heartfelt appreciation to Prof. Arne Holmgren for
recombinant rat TrxR1, and to Prof. Constantinos Koumenis for HEK-TrxR1 and HEK-IRES cells.
The authors confirm that there are no conflicts of interest.
Abbreviations Used
DCFH-DA, 2’,7’-dichlorofluorescin diacetate; DHE, dihydroethidium; DMEM, Dulbecco’s modified
Eagle’s medium; DMSO, dimethyl sulfoxide; DTNB, 5, 5’-dithiobis-2-nitrobenzoic acid; DTT,
DL-dithiothreitol; FBS, fetal bovine serum; FITC, fluorescein 5-isothiocyanate; GR, glutathione
reductase; GSSG, oxidized glutathione; HEK-IRES, HEK cells stably transfected with a control
vector; HEK-TrxR1, HEK cells stably overexpressing TrxR1; HeLa-shNT, HeLa cells stably
transfected with a nontargeting shRNA; HeLa-shTrxR1, HeLa cells stably transfected with
TrxR1-targeting
-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide; PAO, phenylarsenoxide; RIPA
buffer, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% TritonX-100, 0.5% deoxycholate,
.1% SDS, 1 mM PMSF and 1 mM Na VO ; PI, propidium iodide; ROS, reactive oxygen species;
shRNA;
MMP,
mitochondrial
membrane
potential;
MTT,
3
0
3
4
SAN, sanguinarine; Sec, selenocysteine; TCEP, tris(2-carboxyethyl)phosphine; TE buffer, 50 mM
Tris-HCl with 1 mM EDTA, pH 7.5; Trx, thioredoxin; TrxR, thioredoxin reductase.
1
4

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8

SCHEMES, FIGURES AND FIGURE LEGENDS
Scheme 1 Synthesis of three model compounds 5, 6 and 7.
1
9

Figure 1 Inhibition of TrxR by SAN. (A) Chemical structure of SAN. (B) Inhibition of TrxR1,
U498C TrxR1 and GR by SAN. The NADPH-reduced recombinant rat TrxR1 (WT TrxR), U498C
TrxR1, and GR were incubated with the indicated concentrations of SAN for 2 h at room
temperature, then the enzymes activity was determined. (C) Inhibition of TrxR by SAN assayed by
the endpoint insulin reduction assay. The NADPH-reduced TrxR1 was incubated with the indicated
concentrations of SAN for 2 h at room temperature, and the activity was measured by the
Trx-mediated insulin reduction assay. (D) Inhibition of TrxR in HeLa cells. After the cells were
treated with varying concentrations of SAN for 12 h, the enzyme activity of TrxR in HeLa cells was
measured by the endpoint insulin reduction assay. (E) Image-based TrxR activity assay in live HeLa
cells by TRFS-green. HeLa cells were incubated with the indicated concentrations of SAN for 8 h,
and then the TrxR probe TRFS-green was loaded. After the incubation was continued for 4 h, the
fluorescence images were acquired by inverted fluorescence microscopy. Quantification of the
relative fluorescence intensity (RFI) in individual cells was performed by ImageJ. Scale bars: 20 µm.
Data are expressed as mean ± S. E. of three experiments. **, p<0.01 vs. the control group.
2
0

Figure 2 Induction of ROS accumulation of oxidized Trx in HeLa cells. Accumulation of ROS in
HeLa cells by (A) DCFH-DA staining and (B) DHE staining. HeLa cells were treated with various
concentrations of SAN for 6 h followed by incubation with the ROS probe DCFH-DA (10 µM) or
superoxide probe DHE (10 µM) for 30 min. Phase contrast and fluorescence images were acquired
by fluorescence microscopy. Results shown are representative of three independent experiments.
Quantification of the RFI in individual cells was performed by ImageJ. Scale bars: 20 µm. (C)
Measurement of reduced Trx 1 and oxidized Trx 1 by the PAO-sepharose pull down assay. After
treatment of HeLa cells with vehicle or SAN (5 µM) for 12 h, Trx1 redox states were determined. (D)
Quantification of the ratio of reduced Trx1 to oxidized Trx1 by measuring the bands intensity with
ImageJ. **, P< 0.01 vs. the control group.
2
1

Figure 3 Induction of apoptosis by SAN and involvement of TrxR for the cellular actions of SAN.
A) Cytotoxicity of SAN towards HeLa cells. The cells were treated with the indicated
(
concentrations of SAN for 24 or 72 h, and the viability was determined by the MTT assay. (B)
Analysis of apoptosis by Annexin V-FITC/PI double staining assay. HeLa cells were treated with
different concentrations of SAN for 12 h, and the numbers of live cells, apoptotic cells and necrotic
cells were determined by the Annexin V-FITC/PI double staining assay. (C) Activation of caspase-3
by SAN in HeLa cells. HeLa cells were incubated with the indicated concentrations of SAN for 12 h
and the caspase-3 activity in the cell extracts was determined by a colorimetric assay. (D)
SAN-induced disruption of MMP in HeLa cells. After the cells were treated with the indicated
concentrations of SAN for 12 h. the cells were then analyzed by flow cytometry. (E) Cytotoxicity of
SAN on HeLa-shNT and HeLa-shTrxR1 cells. The cells were treated with the indicated
concentrations of SAN for 24 h, and the cell viability was determined by the MTT assay. Inset: the
expression of TrxR1 was analyzed by Western blotting. (F) Cytotoxicity of SAN on HEK-IRES and
HEK-TrxR1 cells. The cells were treated with the indicated concentrations of SAN for 24 h, and the
cell viability was determined by the MTT assay. Inset: the expression of TrxR1 was analyzed by
Western blotting. Data are expressed as mean ± S. E. of three experiments. *P< 0.05; **P< 0.01 vs.
the control groups. Scale bars: 20 µm.
2
2

Figure 4 Phenanthridinium scaffold as a core structure for inhibition of TrxR. (A) Resonance forms
of SAN indicate that the α-carbon next to the nitrogen atom is electrophilic. (B) Inhibition of the
recombinant TrxR by three model compounds.
2
3

HIGHLIGHTS
Sanguinarine (SAN) is a new inhibitor of thioredoxin reductase (TrxR) with an unprecedented
scaffold.
Inhibition of TrxR by SAN promotes oxidative stress-mediated apoptosis.
Inhibition of TrxR by SAN accounts for observed pharmacological functions of SAN.
The phenanthridinium unit is characterized as a new scaffold for designing TrxR inhibitors.