Dual-biomarker-triggered fluorescence probes for differentiating cancer cells and revealing synergistic antioxidant effects under oxidative stress

Article abstract of DOI:10.1039/C8SC03781G

Hydrogen sulfide (H₂S) and human NAD(P)H:quinine oxidoreductase 1 (hNQO1) are potential cancer biomarkers and also vital participants in cellular redox homeostasis. Simultaneous detection of these two biomarkers would benefit the diagnostic precision of related cancers and could also help to investigate their crosstalk in response to oxidative stress. Despite this importance, fluorescent probes that can be activated by the dual action of H₂S detection and hNQO1 activity have not been investigated. To this end, dual-biomarker-triggered fluorescent probes 1 and 2 were rationally constructed by installing two chemoselective triggering groups into one fluorophore. Probe 1 provides a small turn-on fluorescence response toward H₂S but a much larger response to both H₂S and hNQO1 in tandem. By contrast, fluorescence probe 2 is activated only in the presence of both H₂S and hNQO1. Probe 2 exhibits a large fluorescence turn-on (>400 fold), high sensitivity, excellent selectivity as well as good biocompatibility, enabling the detection of both endogenous H₂S and hNQO1 activity in living cells. Bioimaging results indicated that probe 2 could differentiate HT29 and HepG2 cancer cells from HCT116, FHC and HeLa cells owing to the existence of relatively high endogenous levels of both biomarkers. Expanded investigations using 2 revealed that cells could generate more endogenous H₂S and hNQO1 upon exposure to exogenous hydrogen peroxide (H₂O₂), implying the synergistic antioxidant effects under conditions of cellular oxidative stress.

Full text of DOI:10.1039/C8SC03781G

Chemical
Science
View Article Online
View Journal
EDGE ARTICLE
Dual-biomarker-triggered fluorescence probes for
differentiating cancer cells and revealing
synergistic antioxidant effects under oxidative
stress†
Cite this: DOI: 10.1039/c8sc03781g
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
a
b
b
b
*
Changyu Zhang,‡ Qiang-Zhe Zhang,‡ Kun Zhang,‡ Lu-Yuan Li,
c
a
d
* *
Michael D. Pluth,
Long Yi
and Zhen Xi
Hydrogen sulfide (H2S) and human NAD(P)H:quinine oxidoreductase 1 (hNQO1) are potential cancer
biomarkers and also vital participants in cellular redox homeostasis. Simultaneous detection of these two
biomarkers would benefit the diagnostic precision of related cancers and could also help to investigate
their crosstalk in response to oxidative stress. Despite this importance, fluorescent probes that can be
activated by the dual action of H2S detection and hNQO1 activity have not been investigated. To this
end, dual-biomarker-triggered fluorescent probes 1 and 2 were rationally constructed by installing two
chemoselective triggering groups into one fluorophore. Probe 1 provides a small turn-on fluorescence
response toward H2S but a much larger response to both H2S and hNQO1 in tandem. By contrast,
fluorescence probe 2 is activated only in the presence of both H2S and hNQO1. Probe 2 exhibits a large
fluorescence turn-on (>400 fold), high sensitivity, excellent selectivity as well as good biocompatibility,
enabling the detection of both endogenous H2S and hNQO1 activity in living cells. Bioimaging results
indicated that probe 2 could differentiate HT29 and HepG2 cancer cells from HCT116, FHC and HeLa
cells owing to the existence of relatively high endogenous levels of both biomarkers. Expanded
investigations using 2 revealed that cells could generate more endogenous H2S and hNQO1 upon
exposure to exogenous hydrogen peroxide (H2O2), implying the synergistic antioxidant effects under
conditions of cellular oxidative stress.
Received 23rd August 2018
Accepted 9th January 2019
DOI: 10.1039/c8sc03781g
rsc.li/chemical-science
certain cancers can be signicantly improved with early diag-
nosis and treatment.2 Cancer biomarkers are endogenous
Introduction
molecules that are differentially expressed in cancer cells rela-
tive to their normal counterparts. Altered levels of such
biomarkers can be measured to establish a correlation with the
disease process and are useful for cancer diagnosis and
therapy.3 Furthermore, the simultaneous detection of multiple
biomarkers can signicantly increase diagnostic accuracy.4
Recent research has demonstrated that hydrogen sulde (H2S)
and human NAD(P)H:quinine oxidoreductase 1 (hNQO1, EC
1.6.99.2) are potential biomarkers in certain cancer biology,
which suggests that uorescent probes that detect these two
species simultaneously would be of signicant utility.
Cancer, one of the most life-threating diseases, is characterized
as uncontrolled growth and division of normal cells beyond
their natural boundaries. The mortality of cancer remains high,
which is mainly due to metastasis of primary cancer tumors.1
The early stages of cancer development carry the maximum
potential for therapeutic interventions, and the survival rate of
aState Key Laboratory of Organic-Inorganic Composites, Beijing University of Chemical
Technology (BUCT), 15 Beisanhuan East Road, Chaoyang District, Beijing 100029,
China. E-mail: yilong@mail.buct.edu.cn
bState Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai
University, Tianjin 300071, China. E-mail: liluyuan@nankai.edu.cn
As the third endogenous gasotransmitter, H2S is enzymati-
cally generated from cystathionine g-lyase (CSE), cystathionine-
b-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-
MPST)/cysteine aminotransferase (CAT).5 H2S plays important
roles in various biological and pathological progress,6 and
misregulation of endogenous H2S is associated with numerous
diseases.7 Specially, low levels of endogenous H2S appear to
exhibit pro-cancer effects, whereas higher concentrations of H2S
can lead to mitochondrial inhibition and cell death.8 We note
cMaterials Science Institute, Institute of Molecular Biology, Department of Chemistry
and Biochemistry, University of Oregon, Eugene, OR 97403, USA
dState Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, National
Pesticide Engineering Research Center (Tianjin), Collaborative Innovation Center of
Chemical Science and Engineering, Nankai University, China. E-mail: zhenxi@
nankai.edu.cn
† Electronic supplementary information (ESI) available: Experimental details,
photophysical data and some uorescence imaging gures. See DOI:
10.1039/c8sc03781g
‡ These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2019
Chem. Sci.

View Article Online
Chemical Science
Edge Article
that some cancer cells, such as ovarian and colorectal cancer
cell lines, exhibit increased H2S production.9 This increased H2S
may be useful for cell growth and proliferation due to H2S-
induced angiogenesis.9c hNQO1 is a FAD-dependent avopro-
tein that catalyzes the obligatory 2-electron reduction of
quinones to hydroquinones and provides versatile cytopro-
tection with multiple functions.10 Levels of this reductase are
elevated in a number of cancer types, including non-small cell
lung cancer, colon cancer, liver cancer and breast cancer,11
when compared to the surrounding normal tissue, making it an
important cancer biomarker as well as an activator for some
anticancer drugs.12
In addition to their roles as potential cancer biomarkers,
both H2S and hNQO1 are also vital participants in cellular
redox homeostasis. H2S is recognized as a potential antioxi-
dant,13 can reduce disulde bonds, and can react with various
reactive oxygen and nitrogen species. For example, Chang et.
al. reported that vascular endothelial growth factor (VEGF)-
triggered H2S production is dependent on NADPH oxidase-
derived H2O2.14a More recently, we as well as other groups
found that endogenous H2S can be generated upon simulation
of H2O2 through the glutathionylation and subsequent
increased activity of CBS in HEK 293 cells.14b,c In addition,
hNQO1 can reduce ubiquinone and vitamin E quinone to their
active antioxidant forms and can also reduce superoxide to
protect cells during oxidative stress.15 Furthermore, hNQO1
can be an intracellular source of NAD+, which can fuel the
activity of sirtuins to inhibit mitochondrial reactive oxygen
production.16 Despite the importance of H2S and hNQO1 in
these systems, the response of these two biomarkers to
oxidative stress remains largely unknown. To this end, our
goal was to rationally design uorescent probes for simulta-
neous detection of H2S and hNQO1 to provide new chemical
tools for investigating their possible crosstalk in redox
homeostasis.
Results and discussion
Rational design of the dual-biomarker-triggered uorescence
probes
To enable the simultaneous detection of H2S and hNQO1, we
installed two chemoselective trigger groups that respond to H2S
and hNQO1, respectively, into one uorophore. Such dual-
activity probes are superior to traditional single-analyte detec-
tion probes because they provide specic advantages,
including: (1) avoiding inhomogeneous intracellular distribu-
tion from different probes; (2) providing an enhanced off–on
response due to the dual-quenching effects; and (3) enable
a simple method to investigate the cooperative relationship of
the analytes.
To enable access to such dual-responsive probes, we made
use of the trimethyl-lock containing quinone propionic acid
(Q3PA) moiety reported by McCarley's group18a as the triggering
group for hNQO1. For the H2S detection motif, we utilized the
thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) amines,21 which
has been utilized by our group as well as others for development
of excellent H2S probes. Additionally, this H2S sensing motif has
been used for different biological applications including tumor
bioimaging in mice.9c Therefore, we combined the Q3PA and
NBD amine moieties onto coumarin and naphthalimide uo-
rophores to access dual-responsive systems. The Q3PA moiety
can switch off the uorescence of the uorophore by the
photoinduced electron transfer (PET) effect, while the NBD part
can quench the uorescence through the uorescence reso-
nance energy transfer (FRET) effect. We expected that the uo-
rescence of the coumarin and naphthalimide uorophores
would be quenched efficiently from this dual-quenching
strategy, and that only dual activation of both the Q3PA and
NBD motifs would result in uorescence turn-on (Scheme 1).
Synthesis and optical properties of the probes
Recent research has demonstrated that uorescence-based
methods are highly suitable and sensitive for in situ and real-
time visualization of biomolecules.17 Numerous uorescent
probes have been developed for the detection of hNQO1 or H2S
in living systems.18 Until now, however, none of these probes
allows for the simultaneous detection of the chemical (H2S)
and enzymatic (hNQO1) biomarkers via a single probe. To
achieve this goal, we utilized a dual-reactive and dual-
quenching strategy, which we reasoned would improve the
sensitivity and selectivity of the system.19 Dual-activation
probes have recently gained attention due to their ability to
ne-tune responses by requiring the presence of two specic
analytes. For example, Chang et. al. reported the dual-analyte
detection of H2O2 and caspase 8 activity during acute inam-
mation in living mice.20a Similar strategies have also been used
for the successful dual-analyte detection of small mole-
cules.20b–d Herein, we report the rational design and prepara-
tion of H2S and hNQO1 dual-responsive uorescent probes 1
and 2, which were successfully utilized to differentiate cancer
cells and reveal the synergistic antioxidant effects in response
to the oxidative stress.
As outlined in Fig. 1A, the synthesis of probe 1 started from
a formylation reaction to generate 3, which was treated with
dimethyl malonate to form the coumarin derivative 4. Then,
single-reactive probe 6 was synthesized from coupling 4-nitro-7-
piperazinobenzofurazan (NBD-PZ) and the hydrolysis product 5.
Aer N-boc deprotection and further coupling with Q3PA, probe
1 was obtained with relative good overall yield. Probe 2 was
Scheme 1 Schematic illustration of the design for a dual-biomarker-
triggered fluorescent probe, which should only be activated by the
synergistic chemical reaction with H2S and enzymatic reaction with
hNQO1.
Chem. Sci.
This journal is © The Royal Society of Chemistry 2019

View Article Online
Edge Article
Chemical Science
Fig. 2 Time-dependent fluorescent response of probe 1 (1 mM) toward
H2S (200 mM) and/or hNQO1 (1 mg mLꢀ1). (A) 1 was treated with H2S
and hNQO1 simultaneously, or only with H2S (B) or hNQO1 (C). (D)
Time-dependent emissions at 465 nm for 1 treated with hNQO1 and
H2S (black), hNQO1 (green), H2S (red) or probe 1 alone (blue) in PBS
buffer.
Fig. 1 (A) Synthesis route for probe 1. (1) POCl3, DMF, rt, 34%; (2)
dimethyl malonate, piperidine, rt, 84%; (3) THF/10% NaOH ¼ 1 : 1, rt,
then 1 M HCl, 70%; (4) NBD-PZ, EDCI, DMAP, rt, 67%; (5) DCM/TFA ¼ 1/
1, then Q3PA, EDCI, DMAP, rt, 44%. (B) Synthesis route for probe 2. (1)
N-Boc-ethylenediamine, reflux, 65%; (2) piperazine, reflux, 63%; (3)
NBD-Cl, TEA, rt, 66%; (4) DCM/TFA ¼ 1/1, then Q3PA, EDCI, DMAP, rt,
88%.
the absence of analytes (Fig. 2D). Taken together, probe 1 can be
used to detect H2S and hNQO1 in tandem, whereas treatment
with only one of the analytes resulted in a signicantly smaller
response.
prepared from a simple four-step synthesis from commercial
available reagents (Fig. 1B). 4-Bromo-1,8-naphthalic anhydride
was reuxed with N-boc-ethylenediamine to produce 8, aer
which the piperazinyl group was introduced through a nucleo-
philic substitution to form 9. Further reaction with NBD-Cl
afforded 10, which was then deprotected and coupled with
the Q3PA motif to provide probe 2 in good yield. All compounds
were characterized by 1H and 13C{1H} NMR spectroscopy as well
as high-resolution mass spectrometry (HRMS) (see ESI†).
With the probes in hand, we examined the optical response
of 1 toward H2S and hNQO1 in phosphate buffered saline (PBS,
50 mM, pH 7.4 containing 0.007% BSA, 100 mM NADH). As
shown in Fig. S1,† 1 displayed two absorption maxima around
405 nm and 500 nm due to the coumarin and NBD amine
moieties, respectively. Aer reaction with both H2S and hNQO1,
new peaks appeared at 395 and 520 nm, which corresponded to
the production of coumarin uorophore and NBD-SH, respec-
tively.19b Notably, 1 remained water-solubile at concentrations
over 25 mM (Fig. S2†). Prior to activation, 1 was essentially non-
uorescent (F1 ¼ 0.15%) due to the PET-FRET dual-quenching
effect. Aer treatment with both hNQO1 (1 mg mLꢀ1) and H2S
(200 mM) for 2 h, a large increase in emission (220-fold)
appeared at 465 nm (Fig. 2A). When 1 was treated by H2S alone
for 2 h, only a 34-fold uorescence enhancement was observed
(Fig. 2B), which was far lower than the response from hNQO1
and H2S together. When 1 was treated with hNQO1 alone for
2 h, the emission enhancement was negligible (2-fold) (Fig. 2C),
implying a more efficient quenching from the NBD moiety in 1.
Stability investigations showed that 1 was stable in PBS buffer in
To achieve a more efficient single- and dual-quenching
effect, we next assessed the uorescence response of 2 toward
H2S and/or hNQO1. Emission spectra were also recorded in PBS
buffer in the presence of NADH. As shown in Fig. 3A, 2 (F2 ¼
0.041%) was essentially non-uorescent due to the dual-
Fig. 3 Time-dependent fluorescent response of probe 2 (1 mM)
toward H2S (200 mM) and/or hNQO1 (1 mg mLꢀ1). (A) 2 was treated with
H2S and hNQO1 simultaneously, or only with hNQO1 (B) or H2S (C). (D)
Time-dependent emissions at 535 nm for 2 treated with hNQO1 and
H2S (black), hNQO1 (green), H2S (red) or probe 2 alone (blue) in PBS
buffer.
This journal is © The Royal Society of Chemistry 2019
Chem. Sci.

View Article Online
Chemical Science
Edge Article
quenching effect, but a strong emission at 535 nm was observed 500 nm increased (Fig. S5D†), implying that the PET process
when hNQO1 and H2S were added simultaneously. Aer 2 h, the was abolished because the PET effect should result in small
uorescence increase at 535 nm was over 400-fold. Consistent changes in absorbance spectra.22 In addition, probe 2 main-
with our design, treatment of 2 with hNQO1 or H2S alone tained water solubility of over 20 mM under the experimental
resulted in only a negligible uorescence enhancement (3- or 7- conditions (Fig. S6†).
fold, Fig. 3B–D and S3†). When compared with probe 1, we
To gain more detailed information about the sensitivity of
found that probe 2 not only resulted in a larger uorescence the dual-responsive probe, we incubated 2 with different levels
turn-on for combined H2S/hNQO1 activation, but also exhibited of hNQO1 and H2S for 2 h, aer which the emission proles
a lower single-analyte response. Because of these positive were measured. Probe 2 was rst treated with different
properties, we utilized probe 2 for subsequent bioimaging concentrations of H2S (0–200 mM) in the presence of hNQO1 (1
investigations.
mg mLꢀ1). As shown in Fig. 5A and B, the emission at 535 nm
Encouraged by the primary uorescence data, we further was linearly related to the concentrations of H2S from 0 to 75
validated the chemistry associated with the sensing mechanism mM. When added to 1 mg mLꢀ1 hNQO1, a 10 mM H2S solution
by using HRMS and UV-vis analysis. We rst conrmed the resulted in a 46-fold uorescence response. Similarly, we
products of both the single- and dual-analyte reactions of 2 with treated 2 with various levels of hNQO1 (0.2–1 mg mLꢀ1) in the
H2S and/or hNQO1 with HRMS (Fig. 4 and S4†). Product 11 (F3 presence of a constant H2S concentration (50 mM), and observed
¼ 7.0%) of the dual activation reaction was observed as [M + H]+ a uorescence enhancement of 180-fold (Fig. 5C).
325.1652 (calcd for C18H21N4O2+, 325.1659). The hNQO1-
One major requirement for a uorescent probe is that it
triggered product 12 and H2S-triggered product 13 were must exhibit a response toward the targeted analytes but not for
+
observed as [M + H]+ 488.1678 (calcd for C24H22N7O5 , 488.1677) other competing species. In order to conrm that the turn-on
and [M + K]+ 595.2301 (calcd for C32H36KN4O5 , 595.2317), response of 2 was selectively caused by the dual activation of
+
respectively. We did not observe the cross reaction side- hNQO1 and H2S, probe 2 was incubated with different reactive
products (e.g. hNQO1-triggered 13 or H2S-triggered 12) in the sulfur species (SO32ꢀ and S2O32ꢀ), biothiols (Cys, Hcy and GSH)
MS spectra. We next performed UV-vis experiments to further and reactive oxygen species (H2O2 and HClO) in the presence of
probe the reaction mechanism. As shown in Fig. S5A,† the hNQO1 or H2S. As shown in Fig. 5D, only the co-incubation of
absorption spectrum of 2 displayed two maximum absorbance hNQO1 and biothiols could trigger a very slight uorescence
peaks near 350 and 500 nm. Aer treatment with H2S and response (<10-fold, lanes 10–12), which was signicantly lower
hNQO1, both of these peaks disappeared and were replaced by than the response triggered by hNQO1 and H2S (>400-fold, lane
peaks at 400 and 520 nm, which corresponded to the uo-
rophore and NBD-SH, respectively. When H2S alone was added,
new peaks at 400 and 520 nm were also observed (Fig. S5B†).
Furthermore, there was an obvious overlap between the absor-
bance prole of NBD-PZ and the emission prole of 11, indi-
cating an intramolecular FRET effect in probe 2 (Fig. S5C†).
When 2 was treated by hNQO1 alone, the absorbance peak at
Fig. 5 (A) Emission spectra of 2 (1 mM) toward different concentrations
of H2S (0–200 mM) in the presence of hNQO1 (1 mg mLꢀ1). (B) Linear
relationship (R2 ¼ 0.999 up to 75 mM) between the emission at 535 nm
from 2 and the concentration of H2S. (C) Emission spectra of 2 (1 mM)
toward different levels of hNQO1 (0–1 mg mLꢀ1) in the presence of H2S
(50 mM). (D) Emissions at 535 nm of 2 (1 mM) after treatment with
various biologically-relevant species. Lane 0, probe 2 alone; lanes 1–7,
SO32ꢀ (200 mM), S2O32ꢀ (200 mM), Cys (500 mM), Hcy (500 mM), GSH (5
mM), H2O2 (200 mM), HClO (200 mM), respectively, all in the presence
2ꢀ
2ꢀ
of H2S (200 mM); lanes 8–14, SO3 (200 mM), S2O3 (200 mM), Cys
Fig. 4 (A) Chemical structures of products from the single or dual (500 mM), Hcy (500 mM), GSH (5 mM), H2O2 (200 mM), HClO (200 mM),
reactions of 2 with H2S and hNQO1. HRMS spectra of compounds 11 respectively, all in the presence of hNQO1 (1 mg mLꢀ1); lane 15, H2S
(B), 12 (C) and 13 (D).
(200 mM) and hNQO1 (1 mg mLꢀ1).
Chem. Sci.
This journal is © The Royal Society of Chemistry 2019

View Article Online
Edge Article
Chemical Science
15). No uorescence increase was observed when H2O2 or HClO
was added (lanes 6–7 and 13–14). Furthermore, treatment of 2
with dicoumarol, an hNQO1 inhibitor, resulted in a slower
reaction rate than the inhibitor-free controls, conrming the
requirement of hNQO1 for probe activation (Fig. S7†).
Differentiation of cancer cells using the probe 2
We rst evaluated the cytotoxicity of 2 in HT29 cells (human
colorectal epithelial cancer cells) by using the methyl thiazolyl
tetrazolium (MTT) assay. The results showed that aer 2 h of
cellular internalization of 33 mM probe, more than 90% of the
cells remained viable (Fig. S8†), implying a low cytotoxicity of
2. The cytotoxicity of 2 was further studied in HEK293A cells
(human embryonic kidney cells) by monitoring of adherent
cell proliferation through the xCELLigence RTCA system
(Fig. S9†). Compound 2 did not show signicant cytotoxicity
from 0–15 mM aer 24 h incubation, and therefore 10 mM of 2
was used for bioimaging experiments. To investigate whether
2 could be employed to distinguish different types of cancer
cells, several cell types were chosen as model biological
systems. Given the elevated levels of both H2S and hNQO1 in
some colorectal cancer cells, HT29 and HCT116 cells (human
colorectal epithelial cancer cell lines) as well as FHC cells
(human normal colorectal epithelial cell line) were initially
selected.9c Then HepG2 cells (human liver cancer cells) with
Fig. 6 Confocal microscopy images for endogenous H2S and hNQO1
detection in living cells using 2. (A) Cells (ꢁ2 ꢂ 104 cells per well) were
incubated with only 2 (10 mM) for 1 h, washed, then imaged. Scale bar,
10 mm. (B) Relative fluorescence intensity of images from (A). (C)
Relative fluorescence intensity of images from inhibitor-pretreated
a high level of endogenous H2S and HeLa cells (human cervical HT29 cells. N ¼ 3 fields of cells, error bars are means ꢃ sd. *P < 0.05;
**P < 0.01. For (B), the black * was relative to HT29 group, and the red *
was relative to HepG2 group.
cancer cells) with a low level of endogenous H2S were also
introduced.8
We assumed that only the 2-stained cells with relatively high
endogenous levels of both H2S and hNQO1 would display
mM), which is an hNQO1 inhibitor. For the inhibitor-treated
signicant uorescence. Aligned with this expectation, the
groups, HT29 cells were pretreated with the inhibitor for
confocal uorescence images showed clearly differentiable
30 min, then incubated with 2 (10 mM) for 1 h, washed and
responses from the selected cells (Fig. 6A). The uorescence
imaged (Fig. S13†). HT29 cells showed strong uorescence
intensity in HT29 and HepG2 cells was much stronger than that
in other cell lines. The relative uorescence increases in HT29
and HepG2 cells were about 5.3 and 3.7 fold higher than that of
aer incubation with 2 alone for 1 h. In contrast, pretreatment
of one or two inhibitors led to a signicant decrease in uo-
rescence, and the observed uorescence intensity was about
other cells (Fig. 6B). The signicantly different uorescence
a half of that in the group without inhibitors (Fig. 6C). These
observed in cancerous versus non-cancerous cells is consistent
with the probe design and suggests that the probe is differen-
tially activated in cancerous versus non-cancerous cells.
results clearly demonstrated the dual H2S and hNQO1
requirement for 2.
In control experiments for single biomarker detection, two
single-analyte probes NIR-H2S (for H2S detection)9c and NIR-
Investigation of the crosstalk between H2S and hNQO1 under
oxidative stress
hNQO1 (for hNQO1 detection)23 developed by us were separately
incubated with these cells (Fig. S10†). As shown in Fig. S11,†
when cells were treated with NIR-H2S, the HT29, HepG2 and H2O2, a common ROS, was introduced as a stimulus to inves-
HCT116 cells displayed a uorescence response, implying the tigate the potential crosstalk between H2S and hNQO1 in
existence of endogenous H2S in the cells. When cells were cellular redox homeostasis. HeLa cells were selected as the
incubated with NIR-hNQO1, the observed uorescence from the model biological systems due to the relative low levels of the
HT29 and HepG2 cells was stronger than that from the other both endogenous biomarkers. The cells were stained by 2,
three cell lines (Fig. S12†). The results indicated the relatively washed and imaged. As displayed in Fig. 7, 2-stained HeLa cells
high endogenous levels of both H2S and hNQO1 in HT29 and exhibited very weak uorescence. However, a signicant uo-
HepG2 cells, which is consistent with the bioimaging results of rescence response was observed when cells were co-incubated
probe 2.
with 2 and H2O2 (50, 100 or 200 mM) for 1 h. To further
To further conrm the dual-activation of 2 in cancer cells, understand the results, the inhibitors AOAA and dicoumarol
we added aminooxyacetic acid (AOAA, 200 mM), which is an were also used for control experiments (Fig. S14†). The H2O2-
inhibitor of enzymatic H2S synthesis, and dicoumarol (100 stimulated cells displayed a signicant uorescence decrease
This journal is © The Royal Society of Chemistry 2019
Chem. Sci.

View Article Online
Chemical Science
Edge Article
Fig. 7 Confocal microscopy images for concentration-dependent
H2O2-induced fluorescence in living HeLa cells using 2. (A) Cells were
co-incubated with probe 2 (10 mM) and H2O2 (0, 50, 100 or 200 mM) for
1 h, washed and imaged. Scale bar, 10 mm. (B) Relative fluorescence
intensity of images versus H2O2 concentration. N ¼ 3 fields of cells,
error bars are means ꢃ sd. *P < 0.05; **P < 0.01.
when pretreated with one or both inhibitors. The relative
emission (Fig. 8A) showed that the stimulation by H2O2 could
trigger about 3.9-fold uorescence enhancement, which was
much higher than the inhibitor-pretreated control groups
(about 1.8-fold). In addition, aer co-incubation with H2O2
and 2, AOAA-pretreated cells were further treated with Na2S
(150 mM) for 30 min, and a small increase in uorescence was
observed (1.5 fold) when compared with the AOAA-pretreated
control group. These data suggest that endogenous H2S and
hNQO1 could be spontaneously generated in living cells when
cells were suffering from acute oxidative stress caused by
exogenous H2O2.
Based on current knowledge, hNQO1 is regulated by the
Keap1 (Kelch-like ECH-associated protein 1)/Nrf2 (nuclear
factor-erythroid 2-related factor 2)/ARE (antioxidant response
elements) pathway.10 Nrf2 protein levels can rapidly increase in
response to ROS, triggering the expression of hNQO1 to inhibit
the formation of free radicals.15a,24 Meanwhile, elevated Nrf2 can
increase the expression of glutathione reductase (GSR), which
can reduce GSSG to GSH.24d,e Such GSH can be involved in the S-
glutathionylation of CBS under H2O2 to produce CBS-SG, which
would enable more efficient biosynthesis of endogenous
H2S.14b,c Thus, we propose that the synergistic antioxidant effect
of H2S and hNQO1 for handling oxidative stress in living cells is
possibly regulated by Nrf2, which can trigger the expression of
hNQO1 directly and improve endogenous H2S levels indirectly
through controlling GSH (Fig. 8B). Taken together, these results
support a synergistic antioxidant effect under cellular oxidative
stress.
Fig. 8 (A) Relative fluorescence intensity of confocal microscopy
images from H2O2-induced HeLa cells. N ¼ 3 fields of cells, error bars
are means ꢃ sd. *P < 0.05. (B) A proposed mechanism of the syner-
gistic antioxidant effect of H2S and hNQO1 under oxidative stress.
Conclusions
In summary, dual-biomarker-triggered uorescent probes were
developed for the simultaneous detection of two potential
cancer biomarkers. Probe 1 could detect the two biomarkers
with a slight uorescence response toward one biomarker (34-
fold turn-on) and a signicantly enhanced uorescence by dual
activation (220-fold turn-on). By contrast, the uorescence of
probe 2 was signicantly enhanced and showed a greater
response for the dual-activation from H2S and hNQO1 (>400-
fold turn-on). Moreover, probe 2 exhibited high sensitivity,
excellent selectivity and good biocompatibility, which enabled
us to differentiate activation levels in HT29 and HepG2 cells
from FHC, HCT116 and HeLa cells due to the notably different
endogenous levels of H2S and hNQO1 in the cell lines. Impor-
tantly, using the probe 2, we revealed a synergistic antioxidant
effect between H2S and hNQO1 in living cells in response to the
oxidative stress. These results clearly demonstrate the strengths
of this dual reporter system, including the signicant off–on
response, ability to distinguish cancer cells with both cancer
biomarkers, and ability to investigate the crosstalk of analytes.
Chem. Sci.
This journal is © The Royal Society of Chemistry 2019

View Article Online
Edge Article
Chemical Science
We also note, however, potential limitations of this system. For
example, the developed tools only provide information on the
relative levels of the biomarkers in different cell lines rather
than precise quantication measurements. In addition, the
development of probes with longer wavelength emissions would
be needed to translate these systems into more complex
systems, such as animal studies. Based on these needs, we are
currently working to develop related dual-responsive probes
with emission in the near-infrared region for in vivo applica-
tions. Overall, our work has demonstrated the research poten-
tial of dual-responsive uorescent probes in cancer biology and
intracellular redox homeostasis.
5 (a) L. Li, P. Rose and P. K. Moore, Annu. Rev. Pharmacol.
Toxicol., 2011, 51, 169; (b) C. Szabo, Nat. Rev. Drug
Discovery, 2007, 6, 917.
6 (a) G. Yang, L. Wu, B. Jiang, W. Yang, J. Qi, K. Cao, Q. Meng,
A. K. Mustafa, W. Mu, S. Zhang, S. H. Snyder and R. Wang,
Science, 2008, 332, 587; (b) Y.-H. Liu, M. Lu, L.-F. Hu,
P. T.-H. Wong, G. D. Webb and J.-S. Bian, Antioxid. Redox
Signaling, 2012, 17, 141; (c) J. L. Wallace, J. G. P. Ferraz and
M. N. Muscara, Antioxid. Redox Signaling, 2012, 17, 58; (d)
A. Papapetropoulos, A. Pyriochou, Z. Altaany, G. Yang,
A. Marazioti, Z. Zhou, M. G. Jeschke, L. K. Branski,
D. N. Herndon, R. Wang and C. Szabo, Proc. Natl. Acad. Sci.
U. S. A., 2009, 106, 21972.
7 (a) J. L. Wallace and R. Wang, Nat. Rev. Drug Discovery, 2015,
14, 329; (b) L.-F. Hu, M. Lu, P. T. H. Wong and J.-S. Bian,
Antioxid. Redox Signaling, 2011, 15, 405; (c) S. Fiorucci,
E. Antonelli, A. Mencarelli, S. Orlandi, B. Renga, G. Rizzo,
E. Distrutti, V. Shah and A. Morelli, Hepatology, 2005, 42, 539.
8 (a) D. Wu, W. Si, M. Wang, S. Lv and A. Ji, Nitric Oxide, 2015,
50, 38; (b) X. Cao, L. Ding, Z. Xie, Y. Yang, M. Whiteman,
P. K. Moore and J.-S. Bian, Antioxid. Redox Signaling, DOI:
10.1089/ars.2017.7058.
Conflicts of interest
The authors declare no competing nancial interests.
Acknowledgements
This work was supported by NSFC (21572019, 81672747), 111
project (B14004), and the NIH (R01GM113030).
´
9 (a) C. Szabo, C. Coletta, C. Chao, K. Modis, B. Szczesny,
A. Papapetropoulos and M. R. Hellmich, Proc. Natl. Acad.
Sci. U. S. A., 2013, 110, 12474; (b) S. Bhattacharyya, S. Saha,
K. Giri, I. R. Lanza, K. S. Nair, N. B. Jennings,
C. Rodriguez-Aguayo, G. Lopez-Berestein, E. Basal,
A. L. Weaver, D. W. Visscher, W. Cliby, A. K. Sood,
R. Bhattacharya and P. Mukherjee, PLoS One, 2013, 8,
e79167; (c) K. Zhang, J. Zhang, Z. Xi, L.-Y. Li, X. Gu,
Q.-Z. Zhang and L. Yi, Chem. Sci., 2017, 8, 2776.
Notes and references
1 (a) R. L. Siegel, K. D. Miller and A. Jemal, Ca-Cancer J. Clin.,
2018, 68, 7; (b) M. F. Ullah and M. Aatif, Cancer Treat. Rev.,
2009, 35, 193.
2 (a) M. Kalinich and D. A. Haber, Science, 2018, 359, 866; (b)
P. Sidaway, Nat. Rev. Clin. Oncol., 2017, 14, 262; (c)
S. K. Arya and S. Bhansali, Chem. Rev., 2011, 111, 6783; (d)
M. Gao, F. Yu, C. Lv, J. Choo and L. Chen, Chem. Soc. Rev., 10 (a) G. Asher, J. Lotem, L. Sachs, C. Kahana and Y. Shaul, Proc.
2017, 46, 2237; (e) S. J. Skates, Clin. Cancer Res., 2016, 22,
4542.
3 (a) S. M. Hanash, S. J. Pitteri and V. M. Faca, Nature, 2008,
Natl. Acad. Sci. U. S. A., 2002, 99, 13125; (b) A. T. Dinkova-
Kostova and P. Talalay, Arch. Biochem. Biophys., 2010, 501,
116.
452, 571; (b) A. Koch, S. C. Joosten, Z. Feng, T. C. de 11 (a) J. J. Schlager and G. Powis, Int. J. Cancer, 1990, 45, 403; (b)
Ruijter, M. X. Draht, V. Melotte, K. M. Smits, J. Veeck,
J. G. Herman, L. V. Neste, W. V. Criekinge, T. De Meyer
S. Danson, T. H. Ward, J. Butler and M. Ranson, Cancer
Treat. Rev., 2004, 30, 437.
and M. Engeland, Nat. Rev. Clin. Oncol., 2018, 15, 459; (c) 12 (a) P. Liu, J. Xu, D. Yan, P. Zhang, F. Zeng, B. Li and S. Wu,
E. Billatos, J. L. Vick, M. E. Lenburrg and A. E. Spira, Clin.
Cancer Res., 2018, 24, 2984; (d) A. Chevalier, Y. Zhang,
O. M. Khdour, J. B. Kaye and S. M. Hecht, J. Am. Chem.
Chem. Commun., 2015, 51, 9567; (b) W. S. Shin, J. Han,
P. Verwilst, R. Kumar, J. H. Kim and J. S. Kim, Bioconjugate
Chem., 2016, 27, 1419.
Soc., 2016, 138, 12009; (e) H. Zhang, J. Fan, J. Wang, 13 (a) C. Szabo, Nat. Rev. Drug Discovery, 2016, 15, 185; (b)
B. Dou, F. Zhou, J. Cao, J. Qu, Z. Cao, W. Zhao and
X. Peng, J. Am. Chem. Soc., 2013, 135, 17469; (f) Y. Jiao,
J. Yin, H. He, X. Peng, Q. Gao and C. Duan, J. Am. Chem.
B. L. Predmore, D. J. Lefer and G. Gojon, Antioxid. Redox
Signaling, 2012, 17, 119; (c) Y. Kimura and H. Kimura,
FASEB J., 2004, 18, 1165.
Soc., 2018, 140, 5882; (g) K. Gu, Y. Xu, H. Li, Z. Guo, 14 (a) V. S. Lin, A. R. Lippert and C. J. Chang, Proc. Natl. Acad.
S. Zhu, S. Zhu, P. Shi, T. D. James, H. Tian and W.-H. Zhu,
J. Am. Chem. Soc., 2016, 138, 5334; (h) F. Wang, Y. Zhu,
L. Zhou, L. Pan, Z. Cui, Q. Fei, S. Luo, D. Pan, Q. Huang,
R. Wang, C. Zhao, H. Tian and C. Fan, Angew. Chem., Int.
Sci. U. S. A., 2013, 110, 7131; (b) L. Yi, L. Wei, R. Wang,
C. Zhang, J. Zhang, T. Tan and Z. Xi, Chem.–Eur. J., 2015,
21, 15167; (c) W.-N. Niu, P. K. Yadav, J. Adamec and
R. Banerjee, Antioxid. Redox Signaling, 2015, 22, 350.
Ed., 2015, 54, 7349; (i) X. Zhen, J. Zhang, J. Huang, C. Xie, 15 (a) D. Ross and D. Siegel, Curr. Opin. Toxicol., 2018, 7, 67; (b)
Q. Miao and K. Pu, Angew. Chem., Int. Ed., 2018, 57, 7804;
(j) X. Chen, D. Lee, S. Yu, G. Kim, S. Lee, Y. Cho, H. Jeong,
K. T. Nam and J. Yoon, Biomaterials, 2017, 122, 130.
R. E. Beyer, J. Segura-Aguilar, S. Di Bernardo, M. Cavazzoni,
R. Rato, D. Fiorentini, M. C. Galli, M. Setti, L. Landi and
G. Lenaz, Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 2528.
4 (a) C. A. Borrebaeck, Nat. Rev. Cancer, 2017, 17, 199; (b) 16 B. Osborne, N. L. Bentley, M. K. Montgomery and N. Turner,
A. Vargas and C. C. Harris, Nat. Rev. Cancer, 2016, 16, 525.
Free Radicals Biol. Med., 2016, 100, 164.
This journal is © The Royal Society of Chemistry 2019
Chem. Sci.

View Article Online
Chemical Science
Edge Article
17 For selective reviews: (a) X. Chen, F. Wang, J. Y. Hyun, T. Wei,
J. Qiang, X. Ren, I. Shin and J. Yoon, Chem. Soc. Rev., 2016,
45, 2976; (b) D. Wu, A. C. Sedgwich, T. Gunnlaugsson,
L. Yi, Sci. Rep., 2016, 6, 30148; (d) C. Wei, R. Wang,
C. Zhang, G. Xu, Y. Li, Q.-Z. Zhang, L.-Y. Li, L. Yi and Z. Xi,
Chem. – Asian J., 2016, 11, 1376.
E. U. Akkaya, J. Yoon and T. D. James, Chem. Soc. Rev., 20 (a) G. C. Van de Bittner, C. R. Bertozzi and C. J. Chang, J. Am.
2017, 46, 7015; (c) V. S. Lin, W. Chen, M. Xian and
C. J. Chang, Chem. Soc. Rev., 2015, 44, 4596; (d)
M. D. Hartle and M. D. Pluth, Chem. Soc. Rev., 2016, 45,
6108; (e) H. Zhu, J. Fan, J. Du and X. Peng, Acc. Chem. Res.,
2016, 49, 2115.
Chem. Soc., 2013, 135, 1783; (b) A. C. Sedgwick, H.-H. Han,
J. E. Gardiner, S. D. Bull, X.-P. He and T. D. James, Chem.
Sci., 2018, 9, 3672; (c) L. Wu, H.-H. Han, L. Liu,
J. E. Gardiner, A. C. Sedgwick, C. Huang, S. D. Bull,
X.-P. He and T. D. James, Chem. Commun., 2018, 54, 11336;
(d) L. Yuan, W. Lin, Y. Xie, B. Chen and S. Zhu, J. Am.
Chem. Soc., 2012, 134, 1305.
18 (a) W. Ong, Y. Yang, A. C. Cruciano and R. L. McCarley, J. Am.
Chem. Soc., 2008, 130, 14739; (b) W. C. Silvers, B. Prasai,
D. H. Burk, M. L. Brown and R. L. McCarley, J. Am. Chem. 21 (a) L. Yi and Z. Xi, Org. Biomol. Chem., 2017, 15, 3828; (b)
Soc., 2013, 135, 309; (c) S. U. Hettiarachchi, B. Prasai and
R. L. McCarley, J. Am. Chem. Soc., 2014, 136, 7575; (d)
G. G. Dias, A. King, F. Moliner, M. Vendrell and E. N. Silva
C. Wei, L. Wei, Z. Xi and L. Yi, Tetrahedron Lett., 2013, 54,
6937; (c) L. A. Montoya, T. F. Pearce, R. J. Hansen,
L. N. Zakharov and M. D. Pluth, J. Org. Chem., 2013, 78, 6550.
´
Junior, Chem. Soc. Rev., 2018, 47, 12; (e) Y. Qian, J. Karpus, 22 L. Yi, H. Li, L. Sun, L. Liu, C. Zhang and Z. Xi, Angew. Chem.,
O. Kabil, S. Y. Zhang, H. L. Zhu, R. Banerjee, J. Zhao and Int. Ed., 2009, 48, 4034.
C. He, Nat. Commun., 2011, 2, 495; (f) Y. C. Chen, 23 C. Zhang, B.-B. Zhai, T. Peng, Z. Zhong, L. Xu, Q.-Z. Zhang,
C. C. Zhu, Z. H. Yang, J. J. Chen, Y. F. He, Y. Jiao, W. J. He, L.-Y. Li, L. Yi and Z. Xi, Dyes Pigm., 2017, 143, 245.
L. Qiu, J. J. Cen and Z. J. Guo, Angew. Chem., Int. Ed., 2013, 24 (a) P. Nioi and J. D. Hayes, Mutal. Res., 2004, 555, 149; (b)
52, 1688; (g) H. A. Henthorn and M. D. Pluth, J. Am. Chem.
Soc., 2015, 137, 15330.
19 (a) H. Zhang, C. Zhang, R. Liu, L. Yi and H. Sun, Chem.
Commun., 2015, 51, 2029; (b) C. Zhang, L. Wei, C. Wei,
J. Zhang, R. Wang, Z. Xi and L. Yi, Chem. Commun., 2015,
51, 7505; (c) C. Zhang, R. Wang, L. Chen, B. Li, Z. Xi and
J. D. Hayes and M. McMahon, Trends Biochem. Sci., 2009,
34, 176; (c) L. V. Favreau and C. B. Pickett, J. Biol. Chem.,
1993, 268, 19875; (d) K.-A. Jung, B. Choi, C. W. Nam,
M. Song, S. T. Kim, J. Lee and M. K. Kwak, Toxicol. Lett.,
2013, 218, 39; (e) J. D. Hayes and A. T. Dinkova-Kostova,
Trends Biochem. Sci., 2014, 39, 199.
Chem. Sci.
This journal is © The Royal Society of Chemistry 2019