A dithiocarbamate-based H2O2-responsive prodrug for combinational chemotherapy and oxidative stress amplification therapy

Article abstract of DOI:10.1039/c9cc05438c

Here, we report the rational design of a H₂O₂-responsive diethyldithiocarbamate (DTC)-based prodrug, which chelated Cu(ii) to form Cu(DTC)₂ with a high anticancer activity in a tumor-microenvironment and induced oxidative stress amplification, showing superior antitumor toxicity to disulfiram.

Full text of DOI:10.1039/c9cc05438c

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A dithiocarbamate-based H O -responsive
2
2
prodrug for combinational chemotherapy
and oxidative stress amplification therapy†
Cite this: Chem. Commun., 2019,
5, 13896
5
Received 15th July 2019,
Accepted 25th October 2019
a
a
a
b
c
Qingqing Pan, Boya Zhang, Xinyu Peng, Shiyu Wan, Kui Luo,
Wenxia Gao,
d
a
a
Yuji Pu * and Bin He
DOI: 10.1039/c9cc05438c
rsc.li/chemcomm
Here, we report the rational design of a H
dithiocarbamate (DTC)-based prodrug, which chelated Cu(II) to form ability and avoid the limitations such as poor stability and
Cu(DTC) with a high anticancer activity in a tumor-microenvironment tumor selectivity. For example, tumor-microenvironment-
and induced oxidative stress amplification, showing superior anti- sensitive prodrugs could significantly enhance the tumor selec-
2 2
O -responsive diethyl-
A prodrug is an effective strategy to improve DSF’s bioavail-
2
1
7
tumor toxicity to disulfiram.
tivity via stimuli-responsive drug release in tumor tissues.
Reactive oxygen species (ROS) are abundant in various tumors
Disulfiram (DSF) is an oral aldehyde dehydrogenase (ALDH) and have been utilized as an endogenous stimulus to construct
18–21
inhibitor and has been approved by the FDA in the treatment smart ROS-responsive prodrugs and DDSs.
ROS can promote
1
of alcoholism. Recently, its excellent in vitro anticancer activity cell proliferation and differentiation at low concentration yet
has garnered wide attention and it has been explored as a induce apoptosis at high concentration. Because of the elevated
2,3
candidate for drug repurposing in cancer therapy. It is mainly ROS level, cancer cells are under oxidative stress and are more
metabolized to diethyldithiocarbamate (DTC), which could form a susceptible to additional ROS invasion; they adapt to oxidative
complex with Cu(II) to give cancericidal Cu(DTC)
2
(Scheme S1, stress by upregulating antioxidant systems (such as glutathione,
4–7
ESI†).
Although DSF exhibits a broad anticancer activity, its GSH) to maintain the ROS homeostasis. Therefore, a prodrug or
clinical translation is still severely hindered by its super-instability DDS that can trigger both ROS generation and inhibition of the
both in the acidic gastric environment and in the bloodstream, ROS-scavenging system can amplify the intracellular oxidative
compromising the in vivo antitumor efficacy through degradation stress for efficient cancer cell inhibition, fulfilling an oxidative
8
22–26
into metabolites without anticancer activity. To address the stress amplification therapy.
stability issue of DSF, polymer-based drug delivery systems Herein, we developed a H O -responsive prodrug DQ (Fig. 1A)
DDSs) have been exploited to physically load DSF, avoiding that could release DTC and quinone methide (QM) in the
the direct contact with blood and prolonging the half-life of presence of H , which is an important type of ROS present in
However, the inefficient DSF loading (drug loading high concentrations in tumor tissues (50–100 mM). Aryl boronic
contents of DSF are about 2–5%) and potential drug leakage esters were used as H -responsive moieties owing to the ease of
during drug delivery inevitably limit their further clinical synthesis and good biocompatibility.
translation. Recently, Cu(DTC) , rather than DSF, has been to release DTC by a B–C bond-cleavage and a following 1,6-benzyl
2
2
(
2 2
O
9
–12
DSF.
2 2
O
27–29
2 2
DQ was cleaved by H O
2
30–32
directly encapsulated into DDSs for enhanced anticancer elimination.
The DTC chelated Cu(II) to generate Cu(DTC)2
1
3–16
effect.
However, Cu(DTC)2 lacks the selective inhibition with high anticancer activity. The chelation process, like the
ability between cancerous and healthy cells.
reaction of DSF and Cu(II), could induce the elevation of intra-
7,8
cellular ROS. In addition, the released QM exerted as a GSH-
scavenging agent to inhibit the GSH-mediated ROS elimination
a
National Engineering Research Center for Biomaterials, Sichuan University,
33,34
in cancer cells.
The simultaneous ROS generation and GSH
Chengdu 610064, China. E-mail: yjpu@scu.edu.cn
b
depletion cooperated collectively to amplify oxidative stress and
killed cancer cells through apoptosis (Fig. 1B). The DQ prodrug
was demonstrated to be effective for in vitro and in vivo cancer cell
School of Chemical Engineering, Sichuan University, Chengdu 610065, China
c
Huaxi MR Research Center (HMRRC), Department of Radiology,
West China Hospital, Sichuan University, Chengdu 610041, China
d
College of Chemistry & Materials Engineering, Wenzhou University,
inhibition by a combination of Cu(DTC) -based chemotherapy
2
Wenzhou 325027, China
and oxidative stress amplification.
†
Electronic supplementary information (ESI) available: Synthesis and charac-
The prodrug DQ was synthesized by facile reaction of sodium
diethylthiocarbamate and 4-bromomethylphenylboronic acid
terization of prodrug DQ. ROS-responsiveness results of DQ and the in vitro and
in vivo biological results. See DOI: 10.1039/c9cc05438c
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1
Fig. 2 H NMR spectra changes of DQ in the presence (A) and absence (B)
of H . (C) The illustration of the fluorescence competition assay and the
fluorescence changes over time.
2 2
O
2 2
Fig. 1 (A) The proposed H O -responsive mechanism of DQ prodrug to
release QM and DTC. DTC chelates the available Cu(II) to give the
anticancer drug Cu(DTC) . (B) The therapeutic process of the DQ prodrug
2
for combinational chemotherapy and oxidative stress amplification
therapy.
(
Fig. S8, ESI†), high concentration of DQ (12.5 mM) formed
nanoparticles in aqueous solution (DMSO/H O = 0.05%/99.95%,
2
v/v). The nanoparticles limited sufficient contact between H O2
2
and DQ molecules and thus the fluorescence recovery was slow in
pinacol ester in a mixed solvent of methanol and tetrahydro-
2
the beginning. The formation of Cu(DTC) in the fluorescence
furan (Scheme S2, ESI†). The chemical structure of DQ was
1
13
competition assay was further demonstrated by analyzing the
verified by H and C NMR and ESI-MS (Fig. S2–S4, ESI†).
The H -responsiveness of DQ was first investigated by
H NMR (Fig. 2A and Fig. S5, ESI†). The H NMR spectra of DQ
after the treatment of H for different times were obtained
using a mixed solvent of DMSO-d /D O (v/v, 9 : 1). Consistent
with the previous NMR study of benzyl boronic ester-cleavage in
17
mixtures at 12 h via ESI-MS (Fig. S9, ESI†). Together, these
2 2
O
1
1
results validated that DQ could be activated by H O to release
2
2
DTC, which could chelate with available Cu(II) to generate
Cu(DTC)2.
We studied the cytocompatibility of DQ using DSF as a
control. NIH 3T3 cells were incubated with DQ or DSF at varying
concentrations for 24 h; the cell viability was then tested by
an MTT assay (Fig. 3A). DQ showed a much lower cytotoxicity
2 2
O
6
2
3
5
response to H
2
O
2
,
new peaks of 4-hydroxybenzyl alcohol,
which was the addition product of water to QM, were observed
at 6.7 and 7.2 ppm; the peaks at 7.4 and 7.7 ppm were ascribed
to the partly hydrolytic product of boronic acid. Kinetics study
revealed that the reaction was a pesudo-first order reaction with
a half-life of 1.33 h (Fig. S6, ESI†), indicating that DTC could be
(
IC50 4 100 mM) to normal cells than DSF (IC50 of 12.5 mM).
We also investigated the anticancer activity of DSF and DQ against
T1 cancer cells in the presence and absence of Cu(II). DSF or
DQ alone showed low cytotoxicity to 4T1 cancer cells (Fig. 3B).
4
3
5
rapidly released relative to persulfides (7.5 h). In contrast, no
obvious cleavage of DQ was observed in the absence of H
after 7 h and around 28% hydrolysis was observed after 48 h
Fig. 2B and Fig. S7, ESI†), demonstrating the slow degradation
of DQ in the absence of H O . Importantly, there was no
17
However, additional extracellular Cu(II) (1 mM) significantly
enhanced their anticancer activity; the IC50 values of DQ and
DSF in the condition of 1 mM Cu(II) were 0.33 and 1.4 mM,
2 2
O
(
2
2
generation of 4-hydroxybenzyl alcohol in the hydrolysis, suggesting
no release of DTC by mere hydrolysis and thus presumably
displaying low systemic toxicity in a further in vivo study.
To further confirm the H
the formation of Cu(DTC) in the presence of Cu(II), we performed
a fluorescence competition assay.
2 2
O -responsive release of DTC and
2
17,36
As shown in Fig. 2C, the
fluorescence of calcein was quenched by Cu(II) binding. Instant
fluorescence recovery was observed after the addition of excessive
DTC in the DTC group, demonstrating the rapid chelation
between DTC and Cu(II). The DQ group showed time-dependent
Fig. 3 The cell viabilities of NIH 3T3 (A) and 4T1 (B) cells after being
2 2
fluorescence recovery, suggesting the H O -triggered sustained
treated with DSF and DQ at different concentrations. The concentrations
2 2
release of DTC. As shown in the dynamic light scattering results of H O and Cu(II) were 10 and 1 mM, respectively.
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respectively (Fig. 3B). DSF showed a similar cytotoxicity to NIH results (Fig. 4B). Thereafter, the intracellular GSH contents
T3 cells (IC50: 0.25 mM) in the presence of Cu(II) (Fig. 3A). were studied by a GSH–GSSG method. DSF induced negligible
3
However, DQ could induce much less cytotoxicity to NIH 3T3 GSH reduction. However, 1 and 2 mM DQ treatment resulted
cells (IC : 6.58 mM), suggesting higher selectivity to inhibit in a remarkable reduction of the GSH contents of 13.0%
5
0
cancer cells.
and 19.6%, respectively (Fig. 4C), verifying the GSH-depletion
plus Cu(II) could enhance property of QM. Taking together, DQ can be activated by H
and incur oxidative stress amplification of 4T1 cells.
Since apoptosis is the main way of cancer cell death when
cancer cells was much lower than that of tumor tissue and the cells suffer from elevated oxidative level, we then performed a
cell culture medium could consume H . We explored the cell apoptosis study to confirm the oxidative stress amplification.
appropriate H O concentration for in vitro study by an MTT As shown in Fig. 4D and Fig. S12 (ESI†), both DSF and DQ caused
We then studied whether H
2
O
2
2 2
O
the anticancer activity of DQ in 4T1 cells. Exogenous H
2 2
O
was supplemented because the H concentration of in vitro
2 2
O
2 2
O
2
2
assay (Fig. S10, ESI†); the extracellular H O concentration in remarkably enhanced apoptosis (total apoptosis of B60% when
2
2
this study was set to be 10 mM. As shown in Fig. 3B, H O
cells were treated with 1 mM DSF and 2 mM DQ) relative to the
2
2
showed negligible effect on the anticancer activity of DSF
H O
2 2
and Cu(II) treated group (control group). Cu(DTC)
2
showed
(
IC50: 0.40 mM). In contrast, DQ showed significantly enhanced comparable cell apoptosis to the control group, confirming its
14,16
anticancer efficacy (IC50: 0.80 mM) with the assistance of H
2
O
2
anticancer mechanism in a non-apoptosis manner.
A new
and Cu(II). Given that DSF was a dimer of DTC, from the point of compound QAc (chemical structure and NMR result were shown
DTC amounts in DSF and DQ, DQ showed comparable in vitro in Fig. S11, ESI†), which can be activated by H to release QM,
anticancer efficiency to DSF in the presence of H O and Cu(II). was synthesized as a negative control and showed a slight increase
2 2
O
2
2
To explore whether DQ could induce oxidative stress ampli- of cell apoptosis rate. Collectively, we confirmed that DQ could
fication in the presence of H O and Cu(II), we studied the induce high apoptosis rate of 4T1 cells. Although DSF had a
2
2
intracellular ROS level in 4T1 cells by flow cytometry and negligible effect in GSH consumption, it still induced high cell
confocal laser scanning microscopy (CLSM) using DCFH as a apoptosis because of the prompt reaction of DSF and Cu(II) and
probe (Fig. 4A and B). H
2
O
2
+ Cu(II) treated cells were used as their interactions with cancer cells.
The stability of DQ and DSF in blood was then studied by
a control. A H -consuming benzyl boronic ester without DTC-
2 2
O
producing ability (B, the structure was shown in the inset in the monitoring their concentrations in mice blood after intra-
Fig. 4A) was used as a negative control. Cells treated with 1 mM venous administration. The blood concentrations of DQ were
B showed a lower intracellular ROS level than the control group, 5-fold higher than those of DSF in the first 4 h (Fig. S13, ESI†),
confirming the H O -consuming capability. In stark contrast, suggesting the superior biostability of DQ.
2
2
cells treated with 1 mM DSF and DQ showed comparable,
significantly elevated ROS level, which was ascribed to the enhanced in vivo stability of DQ, we further evaluated the in vivo
formation of Cu(DTC) . A higher concentration of DQ (2 mM, anticancer efficacy in the 4T1 tumor-bearing female Balb/C
Encouraged by the excellent in vitro anticancer activity and
2
equivalent to 1 mM DSF) further elevated the intracellular ROS mice. Copper(II) gluconate (CuGlu) was supplemented orally
À1
level. CLSM results further confirmed the intracellular ROS at a dose of 1.5 mg kg in all Cu(II)-involved groups. DSF was
À1
intravenously injected with a dose of 15 mg kg . DQ was
administrated similarly with two different doses (18.5 and
À1
3
7 mg kg in the DQ/Cu(II) and DQ Â 2/Cu(II) groups, which
were respectively half and the same equivalent amount of DTC
to 15 mg kg DSF). The body weights of tumor-bearing mice
À1
were monitored to study the systemic toxicity (Fig. 5A). All the
groups showed a similar trend to the saline group, indicating
the low systemic toxicity of DSF and DQ. The tumor volumes
were monitored and the results are shown in Fig. 5B. The tumor
volumes of the CuGlu group were close to those of the saline
group, manifesting that CuGlu alone had no therapeutic effect.
The DQ Â 2/Cu(II) group showed the best antitumor efficacy
and the DQ/Cu(II) group showed slightly enhanced anticancer
effect towards the DSF/Cu(II) group. The tumor inhibition rates
of the DSF/Cu(II), DQ/Cu(II), and DQ Â 2/Cu(II) groups were
48.1%, 57.5%, and 78.3%, respectively (Fig. S14, ESI†).
Histological analysis was further carried out to assess
the organ toxicity by H&E staining. The cell necrosis in the
DQ Â 2/Cu(II) group was more serious than that in the DSF/
Cu(II) and DQ/Cu(II) groups (Fig. 5C). Moreover, low toxicity to
normal organs was observed in these groups (Fig. S15, ESI†),
suggesting the relatively low organ toxicity of DSF and DQ.
Fig. 4 The intracellular ROS levels of 4T1 cells, determined by flow
cytometry (A) and CLSM (B), after being treated with different formulations.
Relative intracellular GSH contents (C) and cell apoptosis percentages (D)
of 4T1 cells treated with different formulations. (n.s., not significant;
*
, p o 0.05; and **, p o 0.01, n = 3.)
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The ROS-responsive prodrug DQ with copper-dependent cyto-
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This work was supported by the National Natural Science
Foundation of China (No. 51773130 and 51573111), Funda-
mental Research Funds for the Central Universities (YJ201764),
Sichuan Science and Technology Program (2019YJ0051), and
Jiangsu International Cooperation project (BZ2017062). We would
like to thank the Analytical & Testing Center of Sichuan University
for NMR spectroscopy.
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Conflicts of interest
There are no conflicts to declare.
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