Combination of chemotherapy and oxidative stress to enhance cancer cell apoptosis

Article abstract of DOI:10.1039/c9sc05997k

Cancer cells are vulnerable to reactive oxygen species (ROS) due to their abnormal redox environment. Accordingly, combination of chemotherapy and oxidative stress has gained increasing interest for the treatment of cancer. We report a novel seleno-prodrug of gemcitabine (Gem), Se-Gem, and evaluated its activation and biological effects in cancer cells. Se-Gem was prepared by introducing a 1,2-diselenolane (a five-membered cyclic diselenide) moiety into the parent drug Gemvia a carbamate linker. Se-Gem is preferably activated by glutathione (GSH) and displays a remarkably higher potency than Gem (up to a 6-fold increase) to a panel of cancer cell lines. The activation of Se-Gem by GSH releases Gem and a seleno-intermediate nearly quantitatively. Unlike the most ignored side products in prodrug activation, the seleno-intermediate further catalyzes a conversion of GSH and oxygen to GSSG (oxidized GSH) and ROS via redox cycling reactions. Thus Se-Gem may be considered as a suicide agent to deplete GSH and works by a combination of chemotherapy and oxidative stress. This is the first case that employs a cyclic diselenide in prodrug design, and the success of Se-Gem as well as its well-defined action mechanism demonstrates that the 1,2-diselenolane moiety may serve as a general scaffold to advance constructing novel therapeutic molecules with improved potency via a combination of chemotherapy and oxidative stress.

Full text of DOI:10.1039/c9sc05997k

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Combination of chemotherapy and oxidative stress to enhaDnOcI: 1e0.10c39a/Cn9ScC0e5r997K
cell apoptosis
Xinming Li, Yanan Hou, Jintao Zhao, Jin Li, Song Wang, and Jianguo Fang*
State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical
Engineering, Lanzhou University, Lanzhou, Gansu 730000, China
*Corresponding author, Email: fangjg@lzu.edu.cn.
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ABSTRACT
Cancer cells are more vulnerable to reactive oxygen species (ROS) due to their
abnormal redox environment. Accordingly, combination of chemotherapy and oxidative
stress has gained increasing interests for treatment of cancer. We report a novel
seleno-prodrug of gemcitabine (Gem), Se-Gem, and evaluated its activation and biological
effects in cancer cells. Se-Gem was prepared by introducing a 1,2-diselenolane (a
five-membered cyclic diselenide) moiety into the parent drug Gem via a carbamate linker.
Se-Gem is preferably activated by glutathione (GSH) and displays a remarkably higher
potency than Gem (up to a 6-fold increase) to a panel of cancer cells. Activation of Se-Gem
by GSH releases Gem and a seleno-intermediate nearly quantitatively. Unlike the most
ignored side products in the prodrug activation, the seleno-intermediate further catalyzes a
conversion of GSH and oxygen to GSSG (oxidized GSH) and ROS via redox cycling
reactions. Thus Se-Gem may be considered as a suicide agent to deplete GSH and works
by a combination of chemotherapy and oxidative stress. This is the first case that employs
the cyclic diselenide in prodrug design, and the success of Se-Gem as well as its
well-defined action mechanism demonstrates that the 1,2-diselenolane moiety may serve a
general scaffold to advance constructing novel therapeutic molecules with improved potency
via a combination of chemotherapy and oxidative stress.
Keywords: Cyclic diselenide; Prodrug; Glutathione; Reactive oxygen; species; Redox
cycling.
2

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INTRODUCTION
The cellular oxidizing species, generally termed as reactive oxygen species (ROS), are
produced under various physiological processes and involved in many signaling pathways.1,
2 However, excessive accumulation of ROS may lead to oxidative stress that disrupts redox
signaling and causes catastrophic damages to biomolecules.3 To keep the ROS in an
appropriate level, cells are evolutionally adapted diverse antioxidant mechanisms, among
which the glutathione system and thioredoxin system are two principal antioxidant systems
in mammalian cells.4, 5 Cancer cells, due to a complex interplay of genetic aberrations and
multiple misregulated signaling pathways, usually harbor elevated ROS level compared to
their benign counterparts. Accordingly, there are frequently upregulated antioxidant
molecules to balance the ROS and maintain a redox homeostasis in cancer cells. This
abnormal redox environment of cancer cells has set a tone for the ROS-mediated cancer
therapy, i.e., killing cancer cells by increase of ROS production or/and inhibition of cellular
antioxidant network,6, 7 which has been supported by many successful examples.8-13
A general drawback of the most of conventional anticancer drugs is the undesired side
effects, which may be significantly reduced by the prodrug strategy. Prodrugs are inactive
forms of drug molecules, and they are converted to active drugs by various endogenous or
exogenous driving forces. By employing characteristic intracellular and surrounding
environments of cancer cells, prodrugs that could be selectively activated by cancer cells
15
have attracted increasing interests in the past years.14,
Based on the unique
microenvironment of cancer cells, a large number of prodrugs that respond to different
stimuli, such as thiols,16-19 acidic pH,20, 21 ROS,13, 22, 23 enzymes,24, 25 hypoxia26, 27 and
light,28-30 have been disclosed with a great success in the experimental treatment of
various cancer cells. Among various types of prodrugs, exploiting the disulfide scaffold as a
trigger unit has been widely adopted,16-19, 31, 32 as cancer cells usually have higher ability
than normal cells to reduce the disulfide bond due to the elevated level of antioxidant
molecules, such as thioredoxin (Trx), thioredoxin reductase (TrxR) and reduced glutathione
(GSH).33, 34 Compared to the enormous attention on the disulfide unit in the construction of
stimuli-responsive materials,16-19, 35-38, application of the diselenide moiety has remained a
Cinderella field and only limited studies have been reported.31, 39-43
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Depending on the ring size, cyclic disulfides/diselenides suffer from significant ring
tension and display different reactivity from acyclic disulfides/diselenides.44-47 Encouraged
by the recent work employing the cyclic disulfide moiety to construct fluorescent probes and
prodrugs,24, 44, 48-50 we report herein the construction of the seleno-prodrug Se-Gem (Fig. 1)
by introducing a 1,2-diselenolane (five-membered cyclic diselenide) moiety into the
anticancer drug gemcitabine (Gem; Fig. 1), a nucleoside analogue that has been used as a
broad spectrum anticancer agent. The activation and biological effects of Se-Gem in cells
were further evaluated. In contrast to its sulfur analogue S-Gem,24 which is selectively
activated by TrxR and shows lower cytotoxicity than the parent drug Gem, Se-Gem is
preferably activated by GSH and has a remarkably higher cytotoxicity than Gem (up to a
6-fold increase). Mechanistic studies reveal that activation of Se-Gem by GSH released
Gem in a nearly quantitative manner accompanying with the formation of a
seleno-intermediate. More importantly, this seleno-intermediate further catalyzed a
conversion of GSH and oxygen to GSSG (oxidized GSH) and ROS via redox cycling
reactions. A drastic alteration of GSH/GSSG ratio was also observed in cancer cells treated
by Se-Gem. Thus Se-Gem works by a combination of chemotherapy and oxidative stress,
which accounts for the increased potency of Se-Gem compared to Gem. The successful
construction of Se-Gem and clarification of its action mechanism demonstrate that the
1,2-diselenolane scaffold may serve a general building block to advance development of
novel therapeutic molecules that combine chemotherapy and oxidative stress.
RESULTS AND DISCUSSION
Chemical Synthesis. The target molecules were prepared by following the synthetic
routes illustrated in Scheme 1. Hydroxy-substituted cyclic disulfides and diselenides were
prepared as shown in Scheme 1A. The hydroxyl groups of Gem were firstly protected by
reacting with tert-butyldimethylsilyl chloride (TBDMSCl) to provide TBS-Gem (step m,
Scheme 1B), which then reacted with triphosgene and corresponding cyclic alcohols to give
TBS-prodrugs (step n). The masked hydroxyl groups were released by reacting
TBS-prodrugs with tetra-n-butylammonium fluoride (TBAF) in tetrahydrofuran (THF),
affording the final prodrugs (step o). C-Gem and S-Gem were synthesized according to our
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24
previous publication. The chemical structures of the prodrugs are shown in Fig. 1. The
detailed synthetic procedures and characterization of compounds are described in
Supporting Information (SI). Original MS spectra, NMR spectra (Figs. S4-S46) and HPLC
chromatograms are provided in SI (Figs. S47-S53).
Evaluation of Cytotoxicity. The cytotoxicity of different molecules to a panel of tumor
cell lines were tested, and the results are summarized in Table 1. S-Gem was reported as a
TrxR-dependent prodrug, and selectively activated by TrxR. Consistent with our published
results, S-Gem showed less cytotoxicity compared to the parent drug Gem.24 Two control
molecules, i.e., C-Gem and C6-Gem, and S6-Gem were completely inactive under our
experimental conditions (IC50 >10 M). As the free amine group on the dihydropyrimidine
ring of Gem is critical for the efficacy of Gem,51 the inactivation of C-Gem, C6-Gem and
S6-Gem indicates that all of these molecules cannot be activated by cellular environment to
liberate the active drug Gem, which is supported by the following experiments (vide infra).
Interestingly, Se-Gem showed increased cytotoxicity to all the tested cancer cells compared
to Gem, while Se6-Gem had less potency than Gem. We also determined the cytotoxicity of
Se-Gem to three non-cancerous cell lines (Fig. S1 in SI). Interestingly, Se-Gem displays
decreased cytotoxicity to non-cancerous cells (The IC50 values for Se-Gem to L02 cells,
HUVEC cells and 293T cells are 1.09, 2.93 and 0.45 μM, respectively.).
Activation of Prodrugs by GSH and TrxR. As the glutathione system and thioredoxin
system are two major systems to reduce cellular disulfide bonds,52 we then determined the
activation of different molecules by GSH and TrxR. Fig. 2A shows the reduction of prodrugs
by GSH. It is not convenient to determine the direct reduction of the molecules by GSH as
there is no obvious change of absorbance spectra when these molecules were reduced by
GSH. Thus, we adopted a coupled enzymatic assay with glutathione reductase (GR) and
NADPH.24, 44 The decay of absorbance at 340 nm (A340), due to the oxidation of NADPH,
was monitored. Without addition of prodrugs (GSH, NADPH and GR only), the background
rate of NADPH oxidation (rNADPH) is 0.57 M min-1 by taking the 6.22 mM-1 cm-1 as the
extinction coefficient of NADPH. Addition of C-Gem, C6-Gem or S6-Gem did not elicit
significant increase of NADPH oxidation, indicating that GSH could hardly reduce them,
which is consistent with the negligible cytotoxicity of these compounds (Table 1). Addition of
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-1
S-Gem increased the rate of NADPH oxidation slightly (rNADPH =3.37 M min ), suggesting
that S-Gem is a weak substrate of GSH.24 Se6-Gem was also able to be reduced by GSH
(rNADPH =4.03 M min-1). Strikingly, a fast oxidation of NADPH was observed when Se-Gem
was added (rNADPH =35.58 M min-1), indicating that Se-Gem is a good substrate of GSH.
The rate of Se-Gem reduction was also demonstrated to be dependent on GSH
concentrations (Fig 2C). Since Se-Gem was readily reduced by GSH, we next determined
the effect of TrxR in reducing Se-Gem. As shown in Fig. 2B, TrxR has the ability to reduce
Se-Gem. Addition of Trx could further increase the reduction of Se-Gem by TrxR, suggesting
that Se-Gem is also a substrate of Trx. Compared to the fast reduction of Se-Gem by GSH
(rNADPH =35.58 M min-1), the less efficient reduction of Se-Gem by TrxR/Trx (rNADPH =7.00
M min-1, 100 nM TrxR and 10 M Trx) indicates that GSH plays a major role in activation of
Se-Gem. Consistent with our previous results, S-Gem was confirmed to be a good substrate
of TrxR (rNADPH =15.67 M min-1, 50 nM TrxR).24 The results from the reduction of different
molecules by GSH and TrxR also support the conclusion in our recent work, where we
comprehensively studied the reduction of cyclic disulfides and cyclic diselenides by GSH
and TrxR, and disclosed that the five-membered cyclic disulfides are exclusively reduced by
TrxR whereas the five-membered cyclic diselenides can be reduced by both GSH and
TrxR.44 It is worth of noting that the oxidation of NADPH is not stoichiometric when Se-Gem
is a substrate (Fig. 2A & 2B). We further demonstrated that oxygen in the reaction system is
involved in the non-stoichiometric oxidation of NADPH, and a stoichiometric consumption of
NADPH (~100 M) upon reduction of Se-Gem (100 M) was observed under anaerobic
conditions (Fig. 2D). After the anaerobic oxidation of NADPH reached equilibrium (the 40-50
min period of the dot line in Fig. 2D), the rate of NADPH oxidation was determined to be 0.46
M min-1, quite close to the background rate of anaerobic NADPH consumption (0.30 M
min-1). Further introducing air into the reaction system resumed the fast oxidation of NADPH
(rNADPH =23.38 M min-1, the 50-55 min period of the dot line in Fig. 2D).
Process of Gem Release. As GSH displayed much higher efficiency in activating
Se-Gem than did TrxR/Trx, we then determined the release of the active drug Gem in the
presence of GSH. As shown in Fig. 3A, a gradual decrease of Se-Gem (retention time: 12.01
min) and increase of Gem (retention time: 5.87 min) were observed as the incubation time
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increased. Quantification of the time-dependent release of Gem is shown in Fig. 3B.
Intriguingly, we also observed a non-stoichiometric oxidation of GSH to GSSG (retention
time: 4.63 min, Fig. 3A). Under our experimental conditions, we incubated Se-Gem (100 M)
with GSH (5 mM) under air atmosphere, and the GSH was almost quantitatively oxidized to
GSSG (~2.5 mM, Fig. 3C). This is reminiscent of the non-stoichiometric but full oxidation of
NADPH (Fig. 2D). According to the activation mechanisms of S-Gem prodrug by TrxR and
reduction of cyclic diselenides by GSH,24, 53 we reasoned that Se-Gem may be activated in
the similar manner to release Gem and a putative seleno-intermediate (Sel-Int in Scheme
2A). Selenolates are reactive and may transfer electrons to oxygen generating superoxides
and selenyl radicals (Scheme 2B, eq. 1), which further dimerize to form diselenides (eq. 2).
In the presence of a reducing agent, such as GSH, the selenolates are regenerated and
GSH is oxidized to yield GSSG (eq. 3).54 Taken together, selenolates catalyze oxidation of
GSH and generation of superoxides via redox cycling reactions under aerobic conditions
(eq. 4). The proposed selenolate-catalyzed redox cycling reactions were supported by the
aforementioned results, i.e., non-stoichiometric oxidation of NADPH under aerobic
conditions while stoichiometric oxidation of NADPH under anaerobic conditions (Fig. 2D),
and non-stoichiometric oxidation of GSH under anaerobic conditions (Fig.3C). Other
biological thiols, such as Cys, can also activate Se-Gem by the similar pattern as GSH does
yet with less efficiency (Fig. S2 in SI). As GSH is the most abundant thiol in cells, GSH is
expected to play a predominant role in activating Se-Gem in cells.
The presence of a selenolate intermediate was further demonstrated by our recently
developed specific selenolate probe Sel-green.55 The original Se-Gem in the reaction
mixture is 100 M, which should generate 100 M of Sel-Int according to Scheme 2A. A
two-fold dilution of the reaction mixture of Se-Gem (100 M) and GSH (5 mM) was
incubated with Sel-green (10 M) and GSH (1 mM), and the time-dependent increase of the
fluorescence was shown in Fig. 4A. The inset in Fig. 4A shows the fold of fluorescence
increase (F/F0) as the function of incubation times. There is little fluorescence increment if
the same reaction mixture was incubated with Sel-green without GSH (Data not shown). The
putative Sel-Int is not stable in the air, and reacts readily to form a diselenide, which may be
reduced to selenolate in the presence of a reducing agent, e.g., GSH (Scheme 2B). We then
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quantified the selenolate in the reaction mixture of Se-Gem and GSH by constructing a
calibration curve using the authentic diselenide compound diethyl 2,2'-diselanediyldiacetate
(SeW, inset in Fig.4B). As the concentration of SeW rose, the rate of fluorescence increment
increased. The inset in Fig. 4B shows a good linearity between the concentrations of SeW
and the rates of fluorescence increment. Based on the calibration curve, the concentration of
the selenolate in the reaction mixture could be easily obtained as 92 M, quite close to the
theoretical value (100 M).
Since we have demonstrated the presence of a selenolate intermediate in the process of
releasing Gem from Se-Gem, we then present further evidence to show the production of
superoxide upon activation of Se-Gem. It is well known that superoxide may reduce
ferricytochrome c producing ferrocytochrome c, which has a higher absorbance at 550 nm.56
Thus, we employed this assay to detect whether superoxide was produced in the process of
Se-Gem activation. Incubation of Se-Gem with GSH generated a species that may reduce
cytochrome c, which could be inhibited by further addition of superoxide dismutase (SOD)
(Fig. 4C & Fig. 4D). This observation indicates that superoxide was produced when Se-Gem
was incubated with GSH. In contrast, we could not detect the superoxide production by
incubation of S-Gem with GSH (Fig. 4D). Generation of a selenolate intermediate and
superoxide supports the proposed mechanism of Se-Gem activation by GSH (Scheme 2)
and accounts for the observed non-stoichiometric oxidation of NADPH (Fig. 2) and GSH
(Fig. 3) under aerobic conditions. The cellular SOD may then convert the superoxide to
long-lived and cell membrane-permeable hydrogen peroxide (H2O2) that further contributes
to oxidative stress.
To further examine the contribution of Gem and the diselenide unit to the observed
cytotoxicity of Se-Gem, we further compared the cytotoxicity of Se-Gem, Gem and
Se-Toluidine. Fig. S3 (in SI) shows clearly that Se-Toluidine has much less toxicity to all the
tested cell lines than does Se-Gem. This result further supports that the cytotoxicity of
Se-Ge is not solely caused by the cyclic diselenide unit, and both Gem and the diselenide
unit contribute to the increased cytotoxicity of Se-Gem. In addition, as the incorporation of a
strained cyclic disulfide/diselenide moiety into a target molecule could also improve the
molecule’s membrane permeability,45-47 Se-Gem may be uptaken by cells more easily than
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does Gem itself, which could also contribute the increased cytotoxicity of Se-Gem.
Induction of Oxidative Stress and Apoptosis. The biological functions of Se-Gem
were further studied. Treatment of Hep G2 cells with Se-Gem caused a dose-dependent
decrease of the GSH/GSSG ratio and the total cellular thiols (Fig. 5A & Fig. 5B). Although
treatment of the cells with S-Gem also leaded to a significant decrease of the GSH/GSSG
ratio and the total cellular thiols, the effect of Se-Gem was much more pronounced.
Treatment of the cells with Gem gave a slight decrease of the GSH/GSSG ratio, but has a
marginal effect on the total cellular thiols. We also determined the cellular ROS level upon
treatment with different molecules. Two ROS probes, i.e., a general ROS probe 2',
7’-dichlorfluorescein diacetate (DCFH-DA) and a specific superoxide probe dihydroethidium
(DHE), were employed. As shown in Fig. 5C and Fig. 5D, Se-Gem elicited accumulation of
ROS and superoxide in the cells. However, neither S-Gem nor Gem caused elevation of
ROS or superoxide under the same experimental conditions. Taken together, treatment of
Hep G2 cells with Se-Gem causes oxidative stress. Resistance to apoptosis is a general
hallmark of malignant cells, which is caused by a complex interplay of genetic mutations and
various misregulated signaling pathways. We next determined whether Se-Gem could
induce apoptotic cell death of Hep G2 cells by the fluorescein isothiocyanate (FITC)-labeled
Annexin V (Annexin V -FITC) and propidium iodide (PI) double staining assay. The scatter
plots representing the distribution of live cells, apoptotic cells and necrotic cells are shown in
Fig. 6A-D, and quantification of the cell distribution is shown in Fig. 6E. Treatment of the
cells with increasing concentrations of Se-Gem elicited increasing number of apoptotic cells,
while the necrotic cells remained a small amount of all the cells. Thus, Se-Gem
predominantly induces apoptotic cell death of Hep G2 cells.
Chemotherapy, e.g., treatment of cancer by using drugs to kill cancer cells, remains an
important way to deal with cancer, and may be the only treatment to the patients with
metastasized tumors. Elevated generation of ROS and an altered redox homeostasis have
long been observed in various types of cancer cells, and this abnormal redox environment
has been harnessed to kill cancer cells by imposing extra ROS on them through promoting
the cellular ROS production or/and inhibiting the cellular antioxidant system.2, 7 Accordingly,
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the combination of chemotherapy and ROS has gained increasing interests to improve the
anticancer effect of chemotherapeutic drugs.21, 57-60 To keep a constant production of ROS,
quinones, due to their characteristic redox property, have been applied to construct prodrugs
by employing the reducing equivalent NAD(P)H under the catalysis of NAD(P)H quinone
oxidoreductase 1 (NQO1).59, 60 We presented in this work that the redox property of
selenolates could also be harnessed to construct prodrugs that work via combination of
chemotherapy and oxidative stress. In contrast to quinones, which utilize NAD(P)H/NQO1
as reducing partners, selenolates are readily coupled with GSH and oxygen to generate
GSSG and superoxides. Se-Gem was prepared by conjugation of the 1,2-diselenolane unit
to the anticancer drug Gem, and displayed enhanced potency in comparison with the parent
Gem. Further extensive studies revealed that the conjugate Se-Gem was activated
predominantly by GSH to release Gem. More importantly, the seleno-intermediate
generated in the process of Se-Gem activation catalyzed the suicide oxidation of GSH
accompanying with a constant production of ROS. As the most abundant antioxidant
molecule in live cells, GSH is frequently upregulated in cancer cells and essential for the fast
proliferation of cancer cells.4, 34, 61 Thus, targeting GSH inhibition has been considered as an
effective way to kill cancer cells.8, 62 The release of active drug Gem and conversion of
antioxidant GSH to ROS account for the enhanced cytotoxicity of Se-Gem. Accumulation of
GSSG and ROS was also observed in the Hep G2 cells treated with Se-Gem. We also
demonstrated that the cytotoxicity of Se-Gem was mediated by its ability to induce
apoptosis. The successful construction of Se-Gem suggests that conjugation of the
1,2-diselenolane scaffold to anticancer drugs may be a general strategy to prepare novel
therapeutic agents with improved potency.
CONCLUSIONS
In summary, we present a novel strategy with a well-defined mechanism to construct
therapeutic agents that combine chemotherapy and oxidative stress to kill cancer cells.
Conjugation of the 1,2-diselenolane unit to Gem affords Se-Gem, which has enhanced
potency compared to the parent drug Gem. Further mechanistic studies reveal that Se-Gem
is preferably activated by the cellular GSH to liberate the active drug Gem, and causes a
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suicide oxidation of GSH with a simultaneous generation of ROS. Consequently, Se-Gem
displayed an improved potency to induce apoptosis of cancer cells.
ASSOCIATED CONTENT
Supporting Information
The Experimental Section, original spectra (1H NMR, 13C NMR, and MS) and purity analysis
of the synthetic molecules, some supplementary results. This material is available free of
charge via the Internet at www.www.
AUTHOR INFORMATION
Corresponding Author
fangjg@lzu.edu.cn
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Financial supports from the National Natural Science Foundation of China (21778028 &
21572093), the Natural Science Foundation of Gansu Province (18JR4RA003), and the 111
project are acknowledged.
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REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
C. R. Reczek and N. S. Chandel, Curr. Opin. Cell Biol., 2015, 33, 8-13.
K. M. Holmstrom and T. Finkel, Nat. Rev. Mol. Cell. Biol., 2014, 15, 411-421.
H. Sies, C. Berndt and D. P. Jones, Ann. Rev. Biochem., 2017, 86, 715-748.
E. Hatem, N. El Banna and M. E. Huang, Antioxid. Redox Sign., 2017, 27, 1217-1234.
J. Lu and A. Holmgren, Free Radic. Biol. Med., 2014, 66, 75-87.
M. Hornsveld and T. B. Dansen, Antioxid. Redox Sign., 2016, 25, 300-325.
D. Trachootham, J. Alexandre and P. Huang, Nat. Rev. Drug Discov., 2009, 8, 579-591.
H. Ogiwara, K. Takahashi, M. Sasaki, T. Kuroda, H. Yoshida, R. Watanabe, A. Maruyama, H. Makinoshima, F.
Chiwaki, H. Sasaki, T. Kato, A. Okamoto and T. Kohno, Cancer Cell, 2019, 35, 177-190.
X. Sun, W. Wang, J. Chen, X. Cai, J. Yang, Y. Yang, H. Yan, X. Cheng, J. Ye, W. Lu, C. Hu, H. Sun, J. Pu and P. Cao,
Cancer Res., 2017, 77, 926-936.
9.
10.
L. Leanza, M. Romio, K. A. Becker, M. Azzolini, L. Trentin, A. Manago, E. Venturini, A. Zaccagnino, A. Mattarei, L.
Carraretto, A. Urbani, S. Kadow, L. Biasutto, V. Martini, F. Severin, R. Peruzzo, V. Trimarco, J. H. Egberts, C.
Hauser, A. Visentin, G. Semenzato, H. Kalthoff, M. Zoratti, E. Gulbins, C. Paradisi and I. Szabo, Cancer Cell, 2017,
31, 516-531.
11.
I. S. Harris, A. E. Treloar, S. Inoue, M. Sasaki, C. Gorrini, K. C. Lee, K. Y. Yung, D. Brenner, C. B. Knobbe-Thomsen,
M. A. Cox, A. Elia, T. Berger, D. W. Cescon, A. Adeoye, A. Brustle, S. D. Molyneux, J. M. Mason, W. Y. Li, K.
Yamamoto, A. Wakeham, H. K. Berman, R. Khokha, S. J. Done, T. J. Kavanagh, C. W. Lam and T. W. Mak, Cancer
Cell, 2015, 27, 211-222.
12.
D. Trachootham, Y. Zhou, H. Zhang, Y. Demizu, Z. Chen, H. Pelicano, P. J. Chiao, G. Achanta, R. B. Arlinghaus, J.
Liu and P. Huang, Cancer Cell, 2006, 10, 241-252.
13.
14.
J. Noh, B. Kwon, E. Han, M. Park, W. Yang, W. Cho, W. Yoo, G. Khang and D. Lee, Nat. Commun., 2015, 6, 6907.
M. H. Lee, A. Sharma, M. J. Chang, J. Lee, S. Son, J. L. Sessler, C. Kang and J. S. Kim, Chem. Soc. Rev., 2018, 47,
28-52.
15.
16.
17.
X. Zhang, X. Li, Q. You and X. Zhang, Eur. J. Med. Chem., 2017, 139, 542-563.
M. Ye, X. Wang, J. Tang, Z. Guo, Y. Shen, H. Tian and W. H. Zhu, Chem. Sci., 2016, 7, 4958-4965.
M. H. Lee, E. J. Kim, H. Lee, H. M. Kim, M. J. Chang, S. Y. Park, K. S. Hong, J. S. Kim and J. L. Sessler, J. Am. Chem.
Soc., 2016, 138, 16380-16387.
18.
19.
F. Kong, Z. Liang, D. Luan, X. Liu, K. Xu and B. Tang, Anal. Chem., 2016, 88, 6450-6456.
X. Wu, X. Sun, Z. Guo, J. Tang, Y. Shen, T. D. James, H. Tian and W. Zhu, J. Am. Chem Soc., 2014, 136,
3579-3588.
20.
21.
22.
A. Fernandez, M. Vermeren, D. Humphries, R. Subiros-Funosas, N. Barth, L. Campana, A. MacKinnon, Y. Feng
and M. Vendrell, ACS Centr. Sci., 2017, 3, 995-1005.
J. Song, L. Lin, Z. Yang, R. Zhu, Z. Zhou, Z. W. Li, F. Wang, J. Chen, H. Yang and X. Chen, J. Am. Chem. Soc., 2019,
141, 8158-8170.
R. Kumar, J. Han, H. J. Lim, W. X. Ren, J. Y. Lim, J. H. Kim and J. S. Kim, J. Am. Chem. Soc., 2014, 136,
17836-17843.
23.
24.
25.
T. T. Hoang, T. P. Smith and R. T. Raines, Angew. Chem. Int. Edit., 2017, 56, 2619-2622.
X. Li, Y. Hou, X. Meng, C. Ge, H. Ma, J. Li and J. Fang, Angew. Chem. Int. Edit., 2018, 57, 6141-6145.
X. Jia, Y. Zhang, Y. Zou, Y. Wang, D. Niu, Q. He, Z. Huang, W. Zhu, H. Tian, J. Shi and Y. Li, Adv. Mater., 2018, 30,
e1704490.
26.
27.
D. Cui, J. Huang, X. Zhen, J. Li, Y. Jiang and K. Pu, Angew. Chem. Int. Edit., 2019, 58, 5920-5924.
P. Verwilst, J. Han, J. Lee, S. Mun, H. G. Kang and J. S. Kim, Biomater., 2017, 115, 104-114.
12

Page 13 of 22
Chemical Science
28.
29.
V. H. van Rixel, V. Ramu, A. B. Auyeung, N. Beztsinna, D. Y. Leger, S. T. Hilt, S. E. Le DeDveOdI:e1c0,.10T3V.9ieY/wCil9AdrStiCizc,l0e5TO9.n9li7nKe
Betancourt, B. Gildner, T. W. Hudnall, V. Sol, B. Liagre, A. Kornienko and S. Bonnet, J. Am. Chem. Soc., 2019,
141, 18444-18454.
D. W. Zheng, Y. Chen, Z. H. Li, L. Xu, C. X. Li, B. Li, J. X. Fan, S. X. Cheng and X. Z. Zhang, Nat. Commun., 2018, 9,
1680.
30.
31.
X. Q. Wang, F. Gao and X. Z. Zhang, Angew. Chem. Int. Edit., 2017, 56, 9029-9033.
B. Sun, C. Luo, X. Zhang, M. Guo, M. Sun, H. Yu, Q. Chen, W. Yang, M. Wang, S. Zuo, P. Chen, Q. Kan, H. Zhang,
Y. Wang, Z. He and J. Sun, Nat. Commun., 2019, 10, 3211.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
C. Yan, Z. Guo, Y. Shen, Y. Chen, H. Tian and W. H. Zhu, Chem. Sci., 2018, 9, 4959-4969.
J. Zhang, X. Li, X. Han, R. Liu and J. Fang, Trends Pharmacol. Sci., 2017, 38, 794-808.
J. M. Estrela, A. Ortega and E. Obrador, Crit. Rev. Clinic. Lab. Sci., 2006, 43, 143-181.
H. Jia, G. Hu, D. Shi, L. Gan, H. Zhang, X. Yao and J. Fang, Anal. Chem., 2019, 91, 8524-8531.
J. Fu, C. Yu, L. Li and S. Q. Yao, .J. Am. Chem. Soc., 2015, 137, 12153-12160.
M. H. Lee, Z. Yang, C. W. Lim, Y. H. Lee, S. Dongbang, C. Kang and J. S. Kim, Chem. Rev., 2013, 113, 5071-5109.
P. Yuan, X. Mao, X. Wu, S. S. Liew, L. Li and S. Q. Yao, Angew. Chem. Int. Edit., 2019, 58, 7657-7661.
Z. Lou, P. Li, X. Sun, S. Yang, B. Wang and K. Han, Chem. Commun., 2013, 49, 391-393.
S. T. Manjare, S. Kim, W. D. Heo and D. G. Churchill, Org. Lett., 2014, 16, 410-412.
X. Han, F. Yu, X. Song and L. Chen, Chem. Sci., 2016, 7, 5098-5107.
X. Han, X. Song, F. Yu and L. Chen, Chem. Sci., 2017, 8, 6991-7002.
X. He, J. Zhang, C. Li, Y. Zhang, Y. Lu, Y. Zhang, L. Liu, C. Ruan, Q. Chen, X. Chen, Q. Guo, T. Sun, J. Cheng and C.
Jiang, Theranostics, 2018, 8, 4884-4897.
44.
45.
X. Li, B. Zhang, C. Yan, J. Li, S. Wang, X. Wei, X. Jiang, P. Zhou and J. Fang, Nat. Commun., 2019, 10, 2745.
N. Chuard, A. I. Poblador-Bahamonde, L. Zong, E. Bartolami, J. Hildebrandt, W. Weigand, N. Sakai and S. Matile,
Chem. Sci., 2018, 9, 1860-1866.
46.
47.
D. Abegg, G. Gasparini, D. G. Hoch, A. Shuster, E. Bartolami, S. Matile and A. Adibekian, J. Am. Chem. Soc.,
2017, 139, 231-238.
E. Bartolami, D. Basagiannis, L. Zong, R. Martinent, Y. Okamoto, Q. Laurent, T. R. Ward, M. Gonzalez-Gaitan, N.
Sakai and S. Matile, Chem. Eur. J., 2019, 25, 4047-4051.
48.
49.
50.
F. Kong, Y. Zhao, Z. Liang, X. Liu, X. Pan, D. Luan, K. Xu and B. Tang, Anal. Chem., 2017, 89, 688-693.
L. Zhang, D. Duan, Y. Liu, C. Ge, X. Cui, J. Sun and J. Fang, J. Am. Chem. Soc., 2014, 136, 226-233.
G. Butora, N. Qi, W. Fu, T. Nguyen, H. C. Huang and I. W. Davies, Angew. Chem. Int. Edit., 2014, 53,
14046-14050.
51.
J. T. Weiss, J. C. Dawson, C. Fraser, W. Rybski, C. Torres-Sanchez, M. Bradley, E. E. Patton, N. O. Carragher and
A. Unciti-Broceta, J. Med. Chem., 2014, 57, 5395-5404.
52.
53.
54.
55.
56.
57.
C. G. Miller, A. Holmgren, E. S. J. Arner and E. E. Schmidt, Free radical biology & medicine, 2018, 127, 248-261.
Q. Laurent, N. Sakai and S. Matile, Helv. Chim. Acta, 2019, 102, e1800209.
C. W. Nogueira, G. Zeni and J. B. Rocha, Chem. Rev., 2004, 104, 6255-6285.
B. Zhang, C. Ge, J. Yao, Y. Liu, H. Xie and J. Fang, J. Am. Chem. Soc., 2015, 137, 757-769.
T. Liu, J. Zhang, X. Han, J. Xu, Y. Wu and J. Fang, Free Radic. Biol. Med., 2019, 135, 216-226.
S. Wang, G. Yu, Z. Wang, O. Jacobson, L. S. Lin, W. Yang, H. Deng, Z. He, Y. Liu, Z. Y. Chen and X. Chen, Angew.
Chem. Int. Edit., 2019, 58, 14758-14763.
58.
59.
W. Zhang, X. Hu, Q. Shen and D. Xing, Nat. Commun., 2019, 10, 1704.
S. Wang, Z. Wang, G. Yu, Z. Zhou, O. Jacobson, Y. Liu, Y. Ma, F. Zhang, Z. Y. Chen and X. Chen, Adv. Sci., 2019, 6,
1801986.
13

Chemical Science
Page 14 of 22
View Article Online
60.
X. Huang, E. A. Motea, Z. R. Moore, J. Yao, Y. Dong, G. Chakrabarti, J. A. Kilgore, M. A. SilveDrsO,I:P1.0L.1.0P39a/tCid9SaCr,05A9.97K
Cholka, F. Fattah, Y. Cha, G. G. Anderson, R. Kusko, M. Peyton, J. Yan, X. J. Xie, V. Sarode, N. S. Williams, J. D.
Minna, M. Beg, D. E. Gerber, E. A. Bey and D. A. Boothman, Cancer Cell, 2016, 30, 940-952.
E. Desideri, F. Ciccarone and M. R. Ciriolo, Nutrients, 2019, 11, E1926.
61.
62.
M. Zarei, H. Du, A. H. Nassar, R. E. Yan, K. Giannikou, S. H. Johnson, H. C. Lam, E. P. Henske, Y. Wang, T. Zhang,
J. Asara and D. J. Kwiatkowski, J. Exp. Med., 2019, 216, 2635-2652.
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Page 15 of 22
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DOI: 10.1039/C9SC05997K
TABLES, SCHEMES AND FIGURES
Table 1. Cytotoxicity of Gem derivatives a
Compds
HeLa
Hep G2
A549
7721
Gem
3.90±0.41
2.46±0.29
0.69±0.14
0.46±0.10
Se-Gem
S-Gem
0.88±0.18 ** 0.47±0.10 ** 0.11±0.04 ** 0.33±0.08 *
6.52±0.48
>10
2.68±0.31
8.53±0.56
>10
0.85±0.11
3.84±0.29
>10
0.98±0.13
>10
Se6-Gem
C-Gem
C6-Gem
S6-Gem
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
a Compounds were incubated with cells for 96 h, and the cytotoxicity was determined by the MTT assay. The data
represent the half maximal inhibitory concentration (IC50) in micromolar of each compound from triplicates, and
are expressed as mean ± SE from triplicates. *, P < 0.05 and **, P < 0.01 vs. the corresponding Gem groups.
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Chemical Science
Page 16 of 22
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DOI: 10.1039/C9SC05997K
Scheme 1. Synthesis of target molecules
A. Synthesis of cyclic disulfides and diselenides.
B. Synthesis of prodrugs.
HO
X
R
NH2
N
X
R
b
a
c
X
X
O
N
O
O
N
O
NH2
N
KSe
SeK
Cl
Cl
Se
Se
Se
Se
n
o
n
OH
OH
OH
O
O
O
O
HN
N
HN
N
OH
n
n
1,2-diselenolan-4-ol
2
m
N
O
1
N
O
F
F
O
X
O
F
F
O
OH
O
X
f
OH
O
O
e
O
O
d
O
HO
O
F
F
OH
n
O
HO
TBSO
TBSO
OH
O
F
F
O
OH
HO
O
O
h
OH
R
HO
D/L-Tartaric acid
O
5
TBSGem
4
3
HO
TBSO
TBSO
HO
HO
HO
HO
OMs
OMs
HO
HO
O
g
j
Se-TBSGem: R = H, n = 0, X = Se;
C6-TBSGem: R = H, n = 1, X = C;
S6-TBSGem: R = BnO, n = 1, X = S;
Se6-TBSGem: R= BnO, n = 1, X = Se
Se-Gem;
C6-Gem;
S6-Gem;
i
Se
Se
Se
OMs
OMs
Se
O
BnO
Bn-DSTox
Se6-Gem.
6
7
DSTox
HO
HO
Se
Se
BnO
HO
HO
O
p
SH
SH
k
S
S
S
l
S
N
H
O
HO
Bn-DTTox
NCO
DTT
DTTox
p-Tolyl isocyanate
Se-Toluidine
o
o
Reagents and Conditions: (a) N2H4.H2O, Se, 65 C; (b) N2H4.H2O, KOH; (c) HCl, 0 C, Air; (d)
SOCl2, MeOH; (e) TSOH, 2,2-Dimethoxypropane, DCM; (f) NaBH4, MeOH; (g) MSCl, Et3N, DCM; (h)
CH3SO3H, EtOH, reflux; (i) Na/naphthalene/THF, Se; (j) BnBr, KOH, 2-MeTHF,TBAHS; (k) MeOH, KOH,
O2; (l) BrBn, KOH, 2-MeTHF, TBAHS; (m) Imidazole, TBDMSCl, DMF; (n) triphosgene, pyridine, DCM; (o)
TBAF, THF; (p) 1,2-diselenolan-4-ol, pyridine, toluene.
Scheme 2. (A) Activation of Se-Gem by GSH
Se
SG
Se
Se
O
O
NH2
N
Se
HN
O
HN
O
O
N
N
N
O
GSH
GSH
Se
O
F
N
O
F
N
O
F
GSSG
+
F
+
O
O
O
F
F
OH
Se
OH
OH
OH
OH
Se-Gem
OH
Gem
Se-Int
(B) Redox cycling reactions of selenolate, oxygen and GSH
R-Se
(1)
GSSG
O2
O2
R-Se
+
+
R-Se
O2
GSH
(2)
2 R-Se
R-Se-Se-R
GSH GSSG
R-Se-Se-R
O2
R-Se
R-Se-Se-R
R-Se (3)
+
+
(4)
16

Page 17 of 22
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DOI: 10.1039/C9SC05997K
BnO
BnO
Se
Se
S
Se
S
S
S
Se
O
O
O
O
O
O
O
O
O
O
O
O
NH2
Se
Se
O
NH
O
NH
NH
O
NH
O
NH
NH
N
O
N
N
N
N
N
N
N
O
F
F
O
NH
N
O
N
O
F
F
N
O
F
F
N
O
F
F
N
O
F
F
N
O
F
F
F
O
O
O
F
OH
OH
OH
OH
OH
OH
OH
OH
OH
S-Gem
OH
C6-Gem
OH
Se6-Gem
OH
C-Gem
OH
Se-Gem
OH
S6-Gem
Se-Toluidine
Gem
Figure 1. Structure of molecules used in this study.
Figure 2. Reduction of different prodrugs by GSH and TrxR/Trx. (A) Reduction of different
compounds by GSH. NADPH (200 μM), GSH (5 mM) and GR (0.5 U mL-1) were incubated at
o
37 C in TE buffer (50 mM Tris-HCl and 1 mM EDTA, pH 7.4), and the absorbance at 340
nm was monitored. After the mixture was incubated for 5 min, compounds (100 μM) were
added. (B) Reduction of S-Gem and Se-Gem by TrxR/Trx. NADPH (200 μM) and different
amount of TrxR with or without Trx were incubated at 37 oC in TE buffer, and the absorbance
at 340 nm was monitored. After the mixture was incubated for 5 min, compounds (100 μM)
were added. (C) Reduction of Se-Gem by GSH. The reaction conditions were the same as
those described in (A) except the GSH concentrations vary. (D) Aerobic and anaerobic
reduction of Se-Gem by GSH. All experiments were performed in triplicates and the
representative results are shown.
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Chemical Science
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DOI: 10.1039/C9SC05997K
Figure 3. GSH-mediated Gem release from Se-Gem. (A) Se-Gem (100 μM) was incubated
with GSH (5 mM) in TE buffer at 37 °C under air condition, and the reaction mixture was
analyzed by HPLC at the indicated time points. Quantification of the time-dependent release
of Gem and generation of GSSG was shown in (B) and (C). All experiments were performed
in duplicates, and the representative results are shown.
18

Page 19 of 22
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DOI: 10.1039/C9SC05997K
Figure 4. Formation of a selenolate intermediate and production of superoxide in the
process of GSH-mediated Se-Gem activation. (A) Time-dependent fluorescence increase of
the reaction mixture upon incubation with additional GSH (1 mM) and the specific selenolate
probe Sel-green (10 M). The detailed conditions were described in the Experimental
Section. The inset shows the fold of fluorescence increase (F/F0) as the function of
incubation time. (B) Time-dependent increase of fluorescence upon incubation of Sel-green
with varying concentrations of the authentic selenocompound SeW. The structure of SeW
was shown. The inset shows the linearship of the rates of fluorescence increase and the
concentrations of SeW. (C) Production of superoxide in the process of GSH-mediated
Se-Gem reduction. GSH (100 M) and ferric cytochrome c (1 mg mL-1) were incubated in TE
buffer for 6 min, and then Se-Gem (20 M) was added. After the mixture was incubated for
another 8 min, SOD (150 U) was added. The absorbance spectra of the reaction mixture
were recorded every 2 min. (D) Time-dependent change of the absorbance at 550 nm. The
experimental conditions were the same as those described in (C), and the concentration of
S-Gem is 20 M. All experiments were assayed in triplicates, and the representative results
are presented.
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Chemical Science
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DOI: 10.1039/C9SC05997K
Figure 5. Induction of oxidative stress by Se-Gem. (A) Alteration of GSH/GSSG ration in
Hep G2 cells upon different compounds treatment. The cells were treated with indicated
compounds for 72 h, and the intracellular GSH/GSSG ratio was determined. (B) Alteration of
total cellular thiols in Hep G2 cells upon different compounds treatment. The cells were
treated with indicated compounds for 72 h, and the cellular total thiols were determined. (C)
Accumulation of ROS in Hep G2 cells upon Se-Gem treatment. The cells were treated with
different compounds for the indicated times, and cellular ROS level was determined by
DCFH-DA staining. (D) Accumulation of superoxide in Hep G2 cells upon Se-Gem
treatment. The cells were treated with different compounds for the indicated times, and
cellular superoxide level was determined by DHE staining. Scale bars = 25 m.
Quantification of the relative fluorescence intensity (R. F. I.) in individual cells by ImageJ are
shown in (E) and (F). All experiments were assayed in triplicates. The representative results
for (C) and (D) are shown, and others are presented as mean ± SE. In (A) and (B), the
control group were treated without drugs but with the same amount of DMSO (0.1%, v/v). *,
P < 0.05 and **, P < 0.01 vs. the control groups, and ## < 0.01 among different groups. In (E)
and (F), **, P < 0.01 among different groups.
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Page 21 of 22
Chemical Science
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DOI: 10.1039/C9SC05997K
Figure 6. Induction of apoptosis by Se-Gem. (A)-(D) Hep G2 cells were treated with
Se-Gem for 48 h, and the apoptotic cell death was evaluated by the Annex V-FITC/PI double
staining assay. The control group were treated without drugs but with the same amount of
DMSO (0.1%, v/v). (E) Quantification of live cells, apoptotic cells and necrotic cells from the
scatter plots (A)-(D). All experiments were performed in triplicates, and representative
results are shown.
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Chemical Science
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DOI: 10.1039/C9SC05997K
X
O
N
X
O
HO
HN
N
O
O
F
F
X=S, S-Gem
TrxR
N
O
NH2
N
Previous work
HO
F
F
Gemcitabine (Gem)
This work
O
X=Se, Se-Gem
GSH
Gem
R-SeH
+
GSH-dependent activation
Increased potency
OH
OH
GSH + O2
GSSG +
ROS
Gem prodrugs
Convertion of GSH to ROS
Graphical Abstract
22