Selenadiazole derivatives as potent thioredoxin reductase inhibitors that enhance the radiosensitivity of cancer cells

Article abstract of DOI:10.1016/j.ejmech.2014.07.032

Thioredoxin system is an attractive target to overcome radioresistance in cancer therapy. The redox enzyme thioredoxin reductase (TrxR) plays a vital role in restoring cellular thiol redox balance disrupted by radiation-induced reactive oxygens species (ROS) generation and oxidative damage. In this study, a series of 1,2,5-selenadiazoles have been synthesized and identified as highly effective inhibitors of TrxR to disrupt the intracellular redox balance, and thus significantly enhanced the sensitivity of cancer cells to X-ray. Upon irradiation, 1,2,5-selenadiazoles displayed a marked synergistic inhibitory effect on radioresistant A375 melanoma cell through enhancement of ROS overproduction, and subsequent induction of ROS-promoted apoptotic pathways, which triggered then mitochondrial dysfunction and caspase activation, finally resulted in augment of radiotherapeutic efficacy. Interestingly, we also found the interaction sites between 1,2,5-selenadiazole and the model peptide of TrxR, which can be confirmed by MALDI-ToF-MS. These results clearly demonstrate TrxR as a potential target for therapy of radioresistant cancers, and selenadiazole derivatives may be attractive radiosensitizing agent by targeting TrxR.

Full text of DOI:10.1016/j.ejmech.2014.07.032

European Journal of Medicinal Chemistry 84 (2014) 335e342
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Selenadiazole derivatives as potent thioredoxin reductase inhibitors
that enhance the radiosensitivity of cancer cells
,
, *
Yuan-Wei Liang a, Junsheng Zheng b, Xiaoling Li a *, Wenjie Zheng a, Tianfeng Chen a
a Department of Chemistry, Jinan University, Guangzhou 510632, China
b The Third Affiliated Hospital of Sun-Yat-Sen University, Guangzhou 510632, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Thioredoxin system is an attractive target to overcome radioresistance in cancer therapy. The redox
enzyme thioredoxin reductase (TrxR) plays a vital role in restoring cellular thiol redox balance disrupted
by radiation-induced reactive oxygens species (ROS) generation and oxidative damage. In this study, a
series of 1,2,5-selenadiazoles have been synthesized and identified as highly effective inhibitors of TrxR
to disrupt the intracellular redox balance, and thus significantly enhanced the sensitivity of cancer cells
to X-ray. Upon irradiation, 1,2,5-selenadiazoles displayed a marked synergistic inhibitory effect on
radioresistant A375 melanoma cell through enhancement of ROS overproduction, and subsequent in-
duction of ROS-promoted apoptotic pathways, which triggered then mitochondrial dysfunction and
caspase activation, finally resulted in augment of radiotherapeutic efficacy. Interestingly, we also found
the interaction sites between 1,2,5-selenadiazole and the model peptide of TrxR, which can be confirmed
by MALDI-ToF-MS. These results clearly demonstrate TrxR as a potential target for therapy of radio-
resistant cancers, and selenadiazole derivatives may be attractive radiosensitizing agent by targeting
TrxR.
Received 14 March 2014
Received in revised form
6 July 2014
Accepted 10 July 2014
Available online 10 July 2014
Keywords:
Radiosensitization
Selenadiazole
Thioredoxin reductase
ROS
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
attractive drug target. In all thioredoxin system, thioredoxin (Trx)
involves in redox reactions through its conserved active site with
Cancer is the second leading cause of death in the world after
cardiovascular diseases. Today, millions of cancer people extend
their lives attributing to early identification and treatment.
Although radiation therapy is well-known to play an important role
in cancer treatment, the common problem of resistance remains a
major obstacle worthy of exploration [1,2]. Radiation-induced ion-
izations may act directly on the cellular component molecules or
indirectly on water molecules, causing water-derived radicals,
which react with nearby molecules in a very short time, resulting in
oxidation of the affected molecules [3e5]. Reactive oxygen species
(ROS) generated as a result of indirect damage is the principal
mediator of radiation induced damage to biological systems. Gen-
eration of ROS creates oxidative stress and disturbs redox balance
within the cells, triggering up-regulation of antioxidant systems [6]
which conduce to the induction of resistance mechanisms. This
makes the ubiquitous redox enzyme thioredoxin reductase an
two thiol residues (eCyseGlyeProeCyse) which can be reduced by
thioredoxin reductase (TrxR) using electrons from NADPH [7]. Pre-
vious works have showed that 1,2,5-selenadiazole derivatives
exerted extensive biological activities against a wide variety of
cancer cells [8,9], With TrxR-inhibition as the aim, drawing on these
works, three novel benzimidazole group-containing 1,2,5-
selenadiazole-oriented derivatives were designed and synthesized
recently and were subjected to test for inhibition efficiency and
specificity on A375 human melanoma cells, which have traditionally
been considered radiation resistant so that radiation therapy has not
commonly been employed to treat it [10]. In the present study, a
series of 1,2,5-selenadiazole derivatives (Scheme 1) were synthe-
sized and evaluated as in vitro radiosensitizer on the A375 cells as
well as antiproliferation agents. The underlying molecular mecha-
nisms accounting for the synergistic effects were also elucidated.
Chemoradiotherapy (CRT), the concurrent use of radiation
therapy and chemotherapy, is a well-established field that in most
cases works better than just using either of them alone and, actu-
ally contributes to the improvement in the overall health of the
patient as well as extending the life expectancy in some patients
* Corresponding authors.
E-mail address: tchentf@jnu.edu.cn (T. Chen).
http://dx.doi.org/10.1016/j.ejmech.2014.07.032
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

336
Y.-W. Liang et al. / European Journal of Medicinal Chemistry 84 (2014) 335e342
CHO
screened against A375 cells by means of MTT assay (Fig. 1A, B and
C). The results showed that CRT had significantly greater inhibitory
effects than using corresponding radiotherapy or chemotherapy
alone on A375 cells. IC50 value of 2a, 2b and 2c (Fig. 1D) reduced
MeO
OMe
H N
H N
NH
NH
SeO2
OMe
1a
2a, 2b, 2c
OMe
HCl/H2O
DMF/TsOH
OMe
from 28.3
mg/ml, 26.4 mg/ml, 12.4 mg/ml to 9.2 mg/ml, 8.0 mg/ml and
2.2 g/ml (18 h) respectively after combining with 8 Gy. Appar-
m
NH
OMe
OMe
N
Se
N
N
N
Se
ently, the dosage of agents decreased greatly, so that systemic
toxicity was considerably lowered down while the anti-
proliferative activities of the agents were highly enhanced. Agents
have brought great alteration into the IC50 value, especially for 2c,
which in combination with 8 Gy was only approximately one sixth
(1/6) of free 2c. In addition, 2a and 2b, which are structural iso-
merides and have extreme similarity in the chemical structure
bore a remarkably close resemblance to each other in the inhibi-
tory effect on cell viability. Morphological examination of the cells
(Fig. 1E) after corresponding treatment showed the typical feature
of cell death such as cell shrinkage and rounding up of the cells.
Noteworthily, The structure of 2a, 2b or 2c remained the same
after irradiation.
Anticancer drugs exert at least part of their cytotoxic effect by
triggering apoptosis in susceptible cells [15]. Therefore, flow cyto-
metric analysis were performed to determine whether apoptosis
was involved in cell death induced by 1,2,5-selenadiazoles and
corresponding CRT. As shown in Fig. 2A and Sub-G1 cell pop-
ulations (see Supporting information), there was hardly any
apoptosis observed in A375 cells which exposed to 8 Gy alone
without treatment with 2b or 2c, just 2.3%. Nevertheless, treatment
N
N
NH
OMe
2a
1a
2b
OMe
OMe
OMe
OMe
OMe
N
Se
N
N
N
N
Se
N
OMe
OMe
OMe
N
N
H
OMe
2c
OMe
Scheme 1. General method for the synthesis of 1,2,5-selenadiazole derivatives (1a, 2a,
2b and 2c).
[11,12]. Decades ago, researchers introduced a theoretical frame-
work to describe the interaction of cytotoxic chemotherapy and
radiotherapy [13], called “spatial cooperation”, it proposed that the
action of radiation and chemotherapeutic drugs is directed toward
different target sites in the body and might interaction with each
other. Radiation tends to target localized tumors, while chemo-
therapy drugs are likely to be more effective in eliminating
micrometastases. However, relationship between radiotherapy and
chemotherapeutic drugs in biological systems need to be further
elucidated. In this study, a series of 1,2,5-selenadiazoles have been
synthesized and identified as highly effective inhibitors of TrxR to
disrupt the intracellular redox balance, and thus significantly
enhanced the sensitivity of cancer cells to X-ray. Upon irradiation,
1,2,5-selenadiazoles displayed a marked synergistic inhibitory ef-
fect on radioresistant A375 melanoma cell through enhancement of
ROS overproduction, and subsequent induction of ROS-promoted
apoptotic pathways, which triggered then mitochondrial dysfunc-
tion and caspase activation, finally resulted in augment of radio-
therapeutic efficacy. Taken together, these results clearly
demonstrate TrxR as a potential target for therapy of radioresistant
cancers, and selenadiazole derivatives may be attractive radio-
sensitizing agent by targeting TrxR.
with 2b (10
amazingly, in the presence of 8 Gy, this section substantially
increased to 57.6%. Likewise, treatment with 2c (4 g/ml), the
mg/ml) alone only triggered 11.7% of apoptotic cells,
m
proportion of apoptotic cells rose from 36.2% in the absence of 8 Gy
to 57.1% in the presence of 8 Gy. This apparently indicated that in
the presence of 8 Gy, they significantly increased the apoptosis-
inducing effect in comparison with that in the absence of 8 Gy
and definitely the apoptosis is the major mode of cell death induced
by 2b or 2c in the absence or presence of 8 Gy.
2.3. Compounds (2b/2c) in combination with 8 Gy enhance the
apoptosis-inducing effect
There are two main apoptotic pathways: the extrinsic or death
receptor pathway and the intrinsic or mitochondrial pathway [16].
Each requires specific triggering signals to begin an energy-
dependent cascade of molecular events. Since that CRT could
significantly amplify the in vitro anticancer and apoptosis-inducing
effects of 2b or 2c, we subsequently conducted further experiments
to understand the molecular mechanism.
Caspases, closely associated with apoptosis, are aspartate-
specific cysteine proteases and members of the interleukin-
1beta-converting enzyme family [17]. The caspase-cascade sys-
tem plays central roles in the induction, transduction and ampli-
fication of intracellular apoptotic signals. In this study, activation of
two initiator caspases: caspase-8 (Fas/TNF-mediated) caspase-9
(mitochondrial-mediated), and an executor caspase caspase-3
were therefore measured by fluorometric assay for the apoptotic
2. Results and discussion
2.1. Chemistry
In terms of the synthesis of 1,2,5-selenadiazole derivatives, 1a
was synthesized by 3, 3'-Diaminobenzidine (214 mg, 1 mmol) and
selenium dioxide (111 mg, 1 mol) in 0.2 N HCl. Importantly, the
following
condensation
reaction
of
a
and
3,4,5-
Trimethoxybenzaldehyde (molar ratio: 1:1.2) which catalyzed by
p-TSA using DMF as solution with aim of introduction of benz-
imidazoles group ultimately gained three products (Scheme 1). This
is different from previous literatures [14] in terms of classical
condensation reaction of o-phenylenediamine with aldehydes,
which mostly acquired only one or two products. 2a and 2b are
isomerides which were further separated by HPLC (Agilent Edipse
program. Fig. 2B indicated that Exposure of A375 cells to 2c (2 mg/
XDB-C18, 21.2 ꢀ 250 mm, 7
mm) using water and acetonitrile (from
72:28 to 36:64, 8.0 ml/min) as mobile phase and ultraviolet as
detector (320 nm).
ml) alone can increase the activation of caspase-3/8/9, resulted in
the involvement of both intrinsic and extrinsic apoptotic path-
ways. Furthermore, treatment of 2c in the presence of 8 Gy
apparently enhanced the activation caspase-3/8/9 to a higher de-
gree. These results were further confirmed by caspases and
cleavage of PARP as examined by Western blotting. As shown in
Fig. 2C, exposure of A375 cells to 2c in the absence or presence of
8 Gy both caused increase in the activation of caspase-3, caspase-8
and caspase-9, and apparently the latter way was more noticeable,
2.2. In vitro anticancer activities of selenadiazoles in combination
with X-ray
The antiproliferative activities of the 1,2,5-selenadiazole (2a, 2b
and 2c) alone or in combination with X-ray (8 Gy) were firstly

Y.-W. Liang et al. / European Journal of Medicinal Chemistry 84 (2014) 335e342
337
Fig. 1. Compounds 2a, 2b and 2c treatment effects on A375 cell viability in the absence of presence of 8 Gy. (A), (B), (C) Growth inhibition of compounds (2a, 2b, 2c) alone or
combined with 8 Gy on A375 cells. A375 cells were treated increasing concentrations of compounds (2a, 2b, 2c) for 6 h then were exposed or unexposed to 8 Gy irradiation and
further incubated for 12 h. Cell viability was determined by MTT assay. (D) IC50 value of 2a, 2b and 2c. (E) Representative images of A375 cells under different treatments. Cells were
pretreated with individual agent for 6 h, exposed or unexposed to 8 Gy and further incubated for another 12 h. Bars with different characters are statistically different at *P < 0.05.
resulting in induced the proteolytic cleavage of PARP, a protein
serving as a biochemical hallmark of cells undergoing apoptosis.
Treatment of A375 cells with 2c in the presence of 8 Gy led to the
decrease in the level of full-length PARP and substantially with
concomitant increase in the level of cleaved PARP. These results
demonstrated that both the extrinsic and intrinsic pathways were
involved in the CRT (2c þ 8 Gy) treatment-induced apoptosis in
A375 cells.

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Y.-W. Liang et al. / European Journal of Medicinal Chemistry 84 (2014) 335e342
Fig. 2. Induction of cancer cell apoptosis by 2b or 2c alone or in combination with 8 Gy. (A) Flow cytometric analysis of A375 cells exposed to 2b or 2c in the absence or presence of
8 Gy. (B) Activation of Caspase-3/8/9 in A375 cells treated with compound 2c or corresponding CRT for 18 h. (C) Western blot analysis of expression levels of Caspases activation and
PARP cleavage in A375 cells treated with 2c (2 mg/ml) or corresponding CRT for 18 h. Equal loading was confirmed by stripping immunoblots and reprobing for b-actin. All cells were
pretreated with definite concentration of 2b or 2c for 6 h, exposed or unexposed to 8 Gy and further incubated for another 12 h. Significant difference between treatment and
control groups is indicated at P < 0.05 (*) level.
2.4. Compound 2c trigger mitochondria dysfunction in the presence
of 8 Gy
expression. Thus, down-regulation of Bcl-2, up-regulation of Bax
and activation of Bid could be important mechanisms by which 2c
induces mitochondria-mediated apoptosis in A375 cells.
Mitochondria play a crucial role in regulating cell death. Mito-
chondrial membrane potential (Djm) is an important parameter of
mitochondrial function and an indicator of cell health [18]. Deple-
tion of Djm suggests the loss of mitochondrial membrane integrity
reflecting the initiation of the proapoptotic signal, in response to
many mechanistically diverse pro-apoptotic triggers, mitochondria
release multiple pro-apoptotic effectors from their inter-membrane
space. The control and regulation of these apoptotic mitochondrial
events occurs through members of the Bcl-2 family of proteins. The
Bcl-2 family of proteins governs mitochondrial membrane perme-
ability and can be either pro-apoptotic, such as Bax, Bak, Bid, or
anti-apoptotic, such as Bcl-2, Bcl-x, Bcl-XL. These proteins have
special significance since they can determine if the cell commits to
apoptosis or aborts the process. Hence, we examined the effects of
2c in the absence or presence of 8 Gy on the expression levels of
anti-apoptotic and pro-apoptotic Bcl-2 family proteins in A375 cells
by Western blotting. As shown in Fig. 3A, treatments of the cells
with 2c alone increased the total expression levels of the pro-
apoptotic protein Bax and cleavage of Bid, yet decreased the
expression level of anti-apoptotic protein Bcl-2. Moreover, treat-
ment of cells with 2c in the presence of 8 Gy substantially increased
the expression levels of Bax and down-regulated Bid and Bcl-2
2.5. Involvement of MAPKs pathways induced by compound 2c
combined with 8 Gy
The MAPK signaling pathway plays a key role in the regulation
of gene expression, cellular growth, and survival [19]. In this study,
The role of p-JNK and p38 MAPK activities in signaling pathways
induced by 2c alone or combined with 8 Gy were examined by
western blot analysis. As shown in Fig. 3B, exposing A375 cells to 2c
alone can cause a gently increase in p-JNK and p38 in the treated
cells, more importantly, the expression of p-JNK and p38 consid-
erably increased after treated with 2c in the presence of 8 Gy. These
results demonstrated that activation of both p-JNK and p38 play
critical role in the 2c-induced (especially in the presence of 8 Gy)
apoptosis in A375 cells.
To study the role of MARK activation on 2c induced growth in-
hibition, A375 cells were subjected to specific MARK inhibitors
before treated with 2c to examine the effects. The results of MTT
assay showed that pretreatment of SP600125 (JNK inhibitor) did
not have effect on the 2c-induced cell death (Fig. 3C), suggesting
that p-JNK did not play important role in governing cell death
induced by 2c. However, pretreatment with SB258530 (p38

Y.-W. Liang et al. / European Journal of Medicinal Chemistry 84 (2014) 335e342
339
Fig. 3. Effects of 2c in combination with 8 Gy on the expression level of Bcl-2 family proteins and MAPK signaling pathways. (A) Western blot analysis of Bcl-2 family protein. Cells
were pretreated with 4ug/ml 2c for 6 h, exposed or unexposed to 8 Gy and back to another 12 h incubation. (B) The phosphorylation status of JNK and expression levels of p38
MAPK after treatment of 2c alone or combined with 8 Gy. (C) Effects of SP600125 (JNK inhibitor) and SB203580 (p38 inhibitor) on 2c-induced growth inhibition. Cells were
pretreated with 20
mM of different inhibitors 1 h prior to the treatment of 8
mg/ml 2c for 18 h. Cell viability was determined by the MTT assay as described in the experimental
section. (D) Morphology of A375 cells after different treatment.
A
B
Control
8 Gy
Control
8 Gy
g/ml + 8 Gy
g/ml + 8 Gy
2c
2b
200
200
180
160
140
120
100
8
μ
g/ml
g/ml
8 μ
16 μ
8 mg/ml
16 mg/ml
8 mg/ml + 8 Gy
16 mg/ml + 8 Gy
16
μ
180
160
140
120
100
0
30
60
90
120
0
30
60
90
120
Time (min)
Time (min)
C
D
Control
8 Gy
Control
8 Gy
2c
2c
200
180
160
140
120
100
200
180
160
140
120
100
2
4
μ
μ
g/ml
g/ml
2
4
μ
g/ml + 8 Gy
2
μ
μ
g/ml
g/ml
2
4
μ
μ
g/ml + 8 Gy
g/ml + 8 Gy
μg/ml + 8 Gy
4
0
30
60
90
120
0
30
60
90
120
Time (min)
Time (min)
Fig. 4. The role of intracellular ROS generation in A375 cell apoptosis induced by 2b or 2c in the absence or presence of 8 Gy. (A), (B), (C), (D) Intracellular ROS levels measured by
DCF or DHE fluorescence. A375 cells were pretreated with 2b (8 or 16 mg/ml) or 2c (2 or 4 mg/ml) for 6 h, subjected to 0 Gy or 8 Gy X-irradiation, then incubated with H2DCF or DHE
and back to incubation for 30 min at 37 ꢁC.

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Y.-W. Liang et al. / European Journal of Medicinal Chemistry 84 (2014) 335e342
inhibitor) resulted in a marked decline in the proportion of dead
cells. Also, the microscopic examination showed that SB258530
indeed reduced the 2c-induced microscopic changes on cell death,
like cell shrinkage and cell rounding (Fig. 3D). These suggested the
involvement of activation of p38 pathway in the apoptosis of A375
cells induced by 2c.
2.6. ROS-dependent apoptosis induced by cooperative treatment
Reactive oxygen species (ROS), as it has come to be known, is a
collective term that broadly describes O2-derived free radicals,
plays an essential role in cancer cell apoptosis induced by anticar-
cinogenic agents that cause DNA damage in the cell nucleus. Cells
react rapidly to redox imbalance with a plethora of biological re-
sponses, including cell cycle-specific growth arrest, gene tran-
scription, initiation of signal transduction pathways and repair of
damaged DNA. These early events are likely to determine whether a
cell will necrose, senesce, apoptose or survive and proliferate [20].
Therefore, in this study, we investigated ROS generation in the
A375 cells by measuring the DCF and DHE fluorescence intensity.
The results showed that exposing A375 cells to X-ray or 1,2,5-
selenadiazoles alone can trigger ROS generation moderately.
Importantly, combination of X-ray and 1,2,5-selenadiazoles signif-
icantly enhanced 1,2,5-selenadiazoles-induced ROS generation
(Fig. 4). These results reveal that combination of X-ray and 1,2,5-
selenadiazoles can effectively induce cancer cell apoptosis in a
ROS-dependent manner.
Fig. 6. MALDI-TOF-MS of (A) compound 2c, (B) model peptide PMBeAGUVGAGLIK,
and (C) model peptide incubated with 2c at a molar ratio of 1:1 at 37 ꢁC for 18 h. A new
molecular ion peak appears at m/z 892.260 that is attributed to addition of 1 Se atom to
the 8-residue peptide fragment PMBeAGUVGAGL (A8), while another new fragment
peak appears at m/z 389.898 that is due to fragment of compound 2c that loss a Se
atom.
blot analysis was also performed to further examine the expression
level of TrxR and generation of Trx. The results (Fig. 5B) showed
that exposing A375 cell lines to 8 Gy alone caused increase in the
expression level of Trx protein. Nevertheless, there was a significant
decline in the Trx expression when exposed to 2c. In addition,
exposing cells to 2c alone or in combination with 8 Gy witnessed no
dramatic alteration in expression level of TrxR. So, this demon-
strated that 2c-induced inhibition on TrxR might be the major
contributing factor in the cell apoptosis as well as solving the
problem of ionizing radiation induced resistance.
2.7. Compound 2c induced cell apoptosis by targeting TrxR
Thioredoxin reductase (TrxR) system is associated with cancer
cell growth and anti-apoptosis process. TrxR plays a vitally signif-
icant role in regulating cellular redox homeostasis. Ionizing radia-
tion induced alteration in redox homeostasis can up-regulate of
antioxidant systems and conduce to the induction of resistance
mechanisms. This is likely to made TrxR to be a new potential target
[21]. Hence, Aiming to investigate the effects of 2c on redox mod-
ulation by inhibition of TrxR. The insulin reduction assay was per-
formed. As Fig. 5A revealed, exposing A375 cells to 8 Gy can cause
slightly increase in the activities of TrxR, this can subsequently
restore cellular redox equilibrium in A375 cells and thus contribute
to inhibition of oxidative injury, resulting in the radioresistance. On
the contrast, treated cells with 2c significantly decreased TrxR ac-
tivity. This can deter TrxR from providing protection against ROS-
mediated cytotoxicity. Accumulated reactive oxygen species and
dysregulation might ultimately lead to apoptosis in radioresistant
A375 cells. Moreover, 2c in combination with 8 Gy caused
comparatively high decrease in TrxR activity. In addition, Western
2.8. 1,2,5-Selenadiazole-peptide interactions
To obtain further insight on the occurring 1,2,5-selenadiazole
-protein interactions on the molecular level, a MS strategy similar
to that described by Lu et al. [22] was applied relying on MS de-
terminations. Thus, MALDI ToF MS analysis was carried out on pre-
reduced TrxR model peptide PMBeAlaeGlyeSeceValeGlye
AlaeGlyeLeueIleeLys (PMBeAGUVGAGLIK) treated with 2c (1:1
ratio) for 18 h at 37 ꢁC. As shown in Fig. 6AeC, the dramatic decline
in the molecular ion peak of 2c and nearly vanished peak of model
peptide suggest that the interaction between compound 2c and the
model peptide indeed carried out. MS analysis led to a new mo-
lecular ion at m/z 892.26, which corresponds to a single Se atom
assigned to the 8-residue peptide fragment PMBeAGUVGAGL. This
can be further confirmed by the new fragment peak at 389.898,
which was just attributed to loss of a Se atom from compound 2c.
Fig. 5. Alteration of antioxidant enzyme activities of TrxR in A375 cells induced by 2c alone or in combination with 8 Gy. (A) Enzymatic activity of TrxR in A375 cells after treatment
with 2c in the absence or presence of 8 Gy exposure. Cells were pretreated with 2c (2 g/ml) for 6 h, exposed or unexposed to 8 Gy then further incubated for another 12 h. (B) Cell
lysates were subjected to Western blot analysis, protein levels of TrxR and Redox thioredoxin were examined.
m

Y.-W. Liang et al. / European Journal of Medicinal Chemistry 84 (2014) 335e342
341
All these alteration can provide evidence sufficient to support the
medium was carefully aspirated and then replaced with 150
ml/well
claim that leucine residue as the major Se binding site.
of DMSO to solubilize the formazan salt. After shaking for 20 min at
room temperature, the plates were read with a microplate spec-
trophotometer (VersaMax) at 570 nm. The cell proliferation and
viability was expressed as percentage of control (as 100%), each
experiment was repeated on three occasions.
3. Conclusions
Selenadiazole derivatives have been identified as novel anti-
proliferative agents in CRTagainst A375 cancercells, which can result
in distinct apoptosis-inducing activity via mitochondria-mediated,
caspase-dependent and ROS-promoted apoptotic pathways. The
results demonstrated the binding sites between 1,2,5-selenadiazoles
and the model peptide of TrxR, suggesting that 2c can be a potent
agent in CRT for radiation-resistant human melanoma cells by tar-
getting TrxR. This also provide valuable mechanistic clues for the
design and synthesis of selenadiazole-oriented derivatives based on
structureeactivity relationships, giving a direction for further
research on apoptosis-inducing or TrxR-targeting agents.
4.5. Flow cytometric analysis
A375 cells were treated with 2b (10 mg/ml) or 2c (4 mg/ml) for
6 h and then were exposed or unexposed to 8 Gy X-irradiation and
further incubated for another 12 h. After treatments cells were
trypsinized, centrifugated and washed with PBS and went to har-
vest, and fixed in 5 ml ice-cold 75% ethanol overnight at ꢂ20 ꢁC, The
fixed cells were washed with PBS and stained with propidium io-
dide (PI) under dark condition, cells were shaken for 30 min at 37 ꢁC
in the dark [24]. Stained cells were analyzed using a flow cytometer
(Beckman Coulter).
4. Experimental section
4.1. Synthesis of 1,2,3-thiadiazole derivatives (1a, 2a, 2b and 2c)
4.6. Measurement of ROS generation
Commercially available 3, 3'-Diaminobenzidine was utilized as
starting material. A solution of 3, 3'-Diaminobenzidine (214 mg,
1 mmol) and selenium dioxide (111 mg, 1 mol) in 0.2 N HCl (30 ml)
was stirred for 2 h at room temperature. Product was filtered, vac-
uum dried and separated by silica gel and eluted using CH2Cl2/
CH3OH (pure CH2Cl2-1:1) as eluent to gain 1a. Yield: 91%. A mixture
of 1a (290 mg, 1 mmol), p-TSA (17 mg, 0.1 mmol) and 3,4,5-
trimethoxybenzaldehyde (235 mg, 1.2 mol) was dissolved in DMF
(60 ml). The mixture was stirred at 80 ꢁC for 2 h. DMF was evapo-
rated, leaving a residue which was washed with water (3 ꢀ 30 ml),
vacuum dried, monitoring by TLC (GF254, Qingdao Haiyang Chem-
ical Co., Ltd. Qingdao) the residue was separated over silica gel
column using CH2Cl2/CH3OH (pure CH2Cl2-150:1e10:1) as eluent to
afford fraction 1 and compound 2c (yield: 46%). Fraction 1 was
further subjected to reversed-phase HPLC (Agilent Edipse XDB-C18,
The intracellular level of ROS causing by 2b or 2c in A375 cells
was determined using the oxidation-sensitive fluorescent probe
H2DCF and DHE. The cells were cultured in 96-well plates (4 ꢀ 104/
well) for 24 h at 37 ꢁC. Next, A375 cells were treated with different
concentration of 2b (8 or 16
which cells were exposed or unexposed to 8 Gy X-irradiation, then
incubated with 2.5 H2DCF (10 mol/l) or 2.5 DHE (10 mol/l) and
mg/ml) or 2c (2 or 4 mg/ml) for 6 h, after
m
m
m
m
further incubated for another 30 min at 37 ꢁC, protected from light
[25]. After incubation, increase in fluorescence resulting from
oxidation of H2DCF to DCF (485 nm excitation/535 nm emission) or
DHE to hydroethidium (480 nm excitation/610 nm emission) was
measured in a fluorescence microplate reader (Tecan SAFIRE).
Relative fluorescence intensity of treated cells was expressed as
percentage of control (as 100%).
21.2 ꢀ 250 mm, 7
m
m) on a C18 column, using water and acetonitrile
4.7. Activities of TrxR
from 72:28 to 36:64 (8.0 ml/min) as mobile phase and ultraviolet as
detector (320 nm) to gain 2a (yield: 22%) and 2b (yield: 18%).
A375 cells were exposed to 2c (2 mg/ml) alone or in combination
with 8 Gy X-irradiation and TrxR activity was measured using a
Thioredoxin Reductase Assay Kit (Cayman) as per manufacturer's
instructions. In this assay, TrxR catalyzes the reduction of 5, 50-
dithiobis (2-nitrobenzoic) acid (DTNB) with NADPH to 5-thio-2-
nitrobenzoic acid (TNB2ꢂ), which generate a strong yellow color
4.2. Cell culture and irradiation
The human melanoma cells A375 cell line was purchased from
American Type Culture Collection (ATCC, Manassas, VA) and
cultured in DMEM medium, which was enriched with 10% fetal
(
lmax ¼ 412 nm). As other enzymes can also reduce DTNB in crude
bovine serum, 100U/ml penicillin and 100
mg/ml streptomycin. In a
biological samples, hence, TrxR specific inhibitor is utilized to
determine TrxR specific activity.
humidified environment of 5% CO2 atmosphere at 37 ꢁC the cells
were incubated and subcultivated twice a week. Cell lines were
irradiated with 8 Gy at a dose rate of 1.5 Gy/min using an Elekta
Precise linear accelerator machine in room temperature.
4.8. Binding studies in a model peptide
The model peptide PMBeAGUVGAGLIK was incubated with 2c
at a molar ratio of 1:1 at 37 ꢁC for 18 h. MALDI-TOF-MS MS based
assay was carried out as previously reported [26].
4.3. Drug treatment
A375 cells were seeded in 96 well-plates at a density of
4 ꢀ 104 cells, allowed to grow to 70e80% confluence cells and
incubated with different concentrations of 2a, 2b or 2c (0, 1, 2, 4, 8,
4.9. Caspases activities assay
16, 32
m
g/ml) for 6 h, then irradiated with a dose of 0 Gy or 8 Gy X-
Cells (1 ꢀ 106 cells/dish) were seeded in 10 cm dishes. After
ray under ambient conditions and further incubated at 37 ꢁC for
another 12 h (5% CO2).
different treatments with 2c (2 mg/ml) alone or in combination with
8 Gy X-irradiation, cells were collected, washed three times with
PBS and lysed in RIPA buffer, no sooner were Cell lysates clarified by
centrifugation at 12,000 rpm for 5 min than the clear lysates were
measured for protein concentration and caspase activity [27].
4.4. MTT assay
The cell viability was determined by MTT assay as previously
Briefly, total protein (80
then 3 l specific caspase substrates (Ac-DEVD-AMC for caspase-3,
Ac-IETD-AMC for caspase-8 and Ac-LEHD-AMC for caspase-9) were
mg/well) were placed in 96-well plates and
described [23]. Briefly, after 18 h treatment, 30
mg/ml per well of
m
MTT solution (5 mg/ml in PBS) was added and incubated for 4 h, the

342
Y.-W. Liang et al. / European Journal of Medicinal Chemistry 84 (2014) 335e342
added. Plates were incubated at 37 ꢁC for 2 h under dark condition
and caspase activity was determined by fluorescence intensity with
the excitation and emission wavelengths set at 380 and 440 nm
respectively.
Fund for the Doctoral Program of Higher Education of China
(20114401110004) and China Postdoctoral Science Foundation.
Appendix A. Supporting information
4.10. Western blot analysis
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.032.
After different treatments with 2c (2 mg/ml) alone or in com-
bination with 8 Gy X-irradiation to A375 cells, the medium was
aspirated and the cells washed with cold PBS, lysed in ice-cold RIPA
buffer (50 mM TriseHCl, 1% Triton X-100, 150 mM NaCl, 1 mM
EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na3VO4, 0.5%NP-40, 1 mM
PMSF, 10 mg/mL aprotinin, 10 mg/mL leupeptin, pH7.4), Protein
concentrations of cell lysates were determined by a BCA protein
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Science
Foundation
for
Distinguished
Young
Scholars
(S2013050014667) of Guangdong Province, Natural Science Founda-
tion of China (21271002, 21371076, 21201082) and Natural Science
Foundation of Guangdong Province (S2012040006919), Research