Monitoring NAD(P)H by an ultrasensitive fluorescent probe to reveal reductive stress induced by natural antioxidants in HepG2 cells under hypoxia

Article abstract of DOI:10.1039/c9sc02020a

Reductive stress, the opposite of oxidative stress, represents a disorder in the redox balance state which is harmful to biological systems. For decades, the role of oxidative stress in tumor therapy has been the focus of attention, while the effects of reductive stress have been rarely studied. Here, we report the anti-cancer effects of reductive stress induced by three natural antioxidants (resveratrol, curcumin and celastrol). Considering the fact that the solid tumor microenvironment suffers from hypoxia, we performed cell experiments under hypoxic conditions. In order to observe the reductive stress, we first developed an ultrasensitive fluorescent probe (TCF-MQ) for specifically imaging NAD(P)H which is a marker of reductive stress. TCF-MQ responded to NAD(P)H rapidly and exhibited high sensitivity with a detection limit of 6 nM. With the help of TCF-MQ, we found that upon the treatment of HepG2 cells with pharmacological doses of three natural antioxidants under hypoxic conditions, high levels of NAD(P)H were produced before cell death. The excess NAD(P)H resulted in reductive stress instead of oxidative stress. In contrast, under normoxic conditions, there was no reductive stress involved in the process of cell death induced by three natural antioxidants. Therefore, we hypothesize that the mechanism of cancer cell death induced by natural antioxidants under hypoxia should be attributed to the reductive stress.

Full text of DOI:10.1039/c9sc02020a

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Monitoring NAD(P)H by an ultrasensitive fluorescent probe to reveal
reductive stress induced by natural antioxidants in HepG2 cells
under hypoxia
Received 00th January 20xx,
Accepted 00th January 20xx
Xiaohong Pana,b, Yuehui Zhaoa, Tingting Chengb, Aishan Zhenga, Anbin Geb, Lixin Zanga, Kehua Xu
DOI: 10.1039/x0xx00000x
*a, and Bo Tang *a
www.rsc.org/
Reductive stress, the opposite of oxidative stress, represents a disorder in the redox balance state that is harmful to
biological systems. For decades, the role of oxidative stress in tumor therapy has been the focus of attention, while the
effects of reductive stress have been rarely studied. Here, we reported the anti-cancer effects of reductive stress induced by
three natural antioxidants (resveratrol, curcumin and celastrol). Considering that the solid tumor microenvironment suffers
from hypoxia, we performed the cell experiments under hypoxic conditions. In order to observe the reductive stress, we first
developed an ultrasensitive fluorescent probe (TCF-MQ) for specifically imaging NAD(P)H which is a marker of reductive
stress. TCF-MQ responded to NAD(P)H rapidly and exhibited high sensitivity with a detection limit of 6 nM. With the help
of TCF-MQ, we found that pharmacological doses of three natural antioxidants treatment of HepG2 cells under hypoxic
conditions, high levels of NAD(P)H were produced before cell death. The excess NAD(P)H resulted in reductive stress
instead of oxidative stress. In contrast, under normoxic conditions, there was no reductive stress involved in the process of
cell death induced by three natural antioxidants. Therefore, we hypothesize that the mechanism of cancer cell death induced
by natural antioxidants under hypoxia should be attributed to the reductive stress.
.
O2), there is abundant oxygen around the cells, which provides
conditions for the production of reactive oxygen species7. But
this condition is obviously contrary to the hypoxic
microenvironment of solid tumors8,9. Hypoxia is a common
feature in solid tumors. In hepatocellular carcinoma (HCC), the
O2 values in most areas is in the range of 0-10 mmHg (0-
1.32%)10. Therefore, in this study, we revealed a new anti-
cancer mechanism of three natural antioxidants (resveratrol,
curcumin and celastrol) under the simulated tumor hypoxic
conditions.
Introduction
Natural antioxidant, such as resveratrol, curcumin or celastrol,
is a self-protective substance produced by organisms to resist
poor environment and reactive oxygen species (ROS). It mainly
exists in fruits, vegetables and medicinal plants. Due to the
antioxidant and free radical scavenging effects, natural
antioxidant is traditionally used as food protection and disease
prevention. Recently, several natural antioxidants have been
reported to possess anti-tumor effects1-3, but the mechanisms
underlying their anticancer functions are debated. Most studies
suggest that some natural antioxidants can stimulate the
production of ROS to cause oxidative stress, which leads to cell
apoptosis4-6. However, these results are contradictory to their
antioxidant effects. We found that there is a common feature in
the above studies, that is, the tumor cells used in the
experiments are all cultured under normoxic conditions (20%
The redox balance in the body is the fundamental to sustain
life. Once redox imbalanced, cell damage and apoptosis are
inevitable11-13. For decades, studies on the anti-tumor effects of
natural antioxidants have been focused on the oxidative stress.
However, reductive stress, another way of redox imbalance, is
rarely noticed. Similar to oxidative stress, reductive stress also
represents a disturbance in the redox state which can cause
body damage and lead to cell death14-16. Recently, reductive
stress is defined as an excessive amount of reducing equivalents,
and excess NAD(P)H and/or GSH is considered a marker of
a. College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory
of Molecular and Nano Probes, Ministry of Education, Collaborative Innovation C
enter of Functionalized Probes for Chemical Imaging in Universities of Shandong, S
handong Normal University, Jinan 250014, P. R. China.
reductive stress14,17,18
.
NAD(P)H, the general term of the nicotinamide adenine
dinucleotide (NAD) derivatives NADH and NADPH19, is
universal electron carrier in cellular processes. The NAD(P)H
and NAD(P)+ undergo reversible oxidation-reduction in many
biological reactions20, and the level of NAD(P)H is tightly
b. Department of Pharmaceutical Sciences, Binzhou Medical University, Yantai
264003, P. R. China.
E-mail: tangb@sdnu.edu.cn; xukehua@sdnu.edu.cn.
†Electronic Supplementary Information (ESI) available:
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linked to the redox environment in cells. Insufficient NAD(P)H 13C NMR, and HRMS (see Experimental VSieewcAtiroticnle Oanlninde
DOI: 10.1039/C9SC02020A
induces oxidative stress due to increased ROS production, Supporting Information).
while excessive NAD(P)H leads to reductive stress because it
cannot be properly oxidized20. Besides, NAD(P)H is a highly Scheme 1. Synthesis of TCF-MQ
reducible dynamic molecule with very high reactivity and
instability that cannot be easily detected in cells and animal
models. Therefore, an ultrasensitive and rapid method to
selectively determine NAD(P)H in vivo must be developed to
investigate the reductive stress.
Fluorescent probe offers
a powerful tool for direct
visualization of biomolecules in vivo with high sensitivity and
short response times21,22. To date, detection methods for
reductive stress have been reported23-25, and in particular,
several small-molecule fluorescent probes for detecting
NAD(P)H have been developed26-31. Louie et al. synthesized a
NADH-sensitive
multimodal
magnetic-resonance/optical-
imaging contrast agent and successfully applied it in cells27.
Komatsu reported an ubiquinone-rhodol-derived fluorescent
probe for imaging intracellular NAD(P)H29. Recently, Chang et
al. developed
a boronic acid-containing fluorescent probe for
NADH based on resazurin, which shows dramatically improved
sensitivity (detection limit: 0.084 μM) by introducing boronic
acid to bind with NADH31. In addition, our group previously
developed a near-infrared small-molecule fluorescent probe
(DCI-MQ) based on dicyanoisophorone for monitoring of
NAD(P)H in cells32. This probe was successfully used to
The proposed recognition mechanism of TCF-MQ toward
NAD(P)H is shown in Scheme 2. The active hydrogen atom of
NAD(P)H was added to the quinolinium moiety of the probe to
form the adduct. To verify the sensing mechanism of TCF-MQ
for NADH, the probe TCF-MQ and the product of TCF-MQ
with NADH were analyzed by ESI-HRMS and 1H NMR
spectroscopy. Once the reaction of TCF-MQ with NADH was
completed, a mass peak at 353.1477 (m/z calcd = 353.1396)
belonging to the reaction product was observed (Scheme 2, Fig.
S1). From the 1H NMR analysis, after the reaction of TCF-MQ
with NADH, the N-CH3 protons of TCF-MQ upfield shifted
from 4.68 to 3.54 ppm, and a new singlet at 3.87 ppm emerged
(Fig. S2). These data confirmed our proposed recognition
mechanism.
selectively detect the endogenous NAD(P)H in cells with
a
detection limit of 12 nM. Although the above methods for
detecting NAD(P)H are suitable for cell use, these methods are
not suitable for monitoring the reductive stress due to their
lower sensitivity.
Here, we designed and synthesized an ultrasensitive small-
molecule fluorescent probe (TCF-MQ) for better detecting
NAD(P)H. TCF-MQ chose
a
more electron-absorbing
tricyanofuran as the chromophore which enables the probe has
good sensitivity for NAD(P)H with the limit of detection (LOD)
was 6 nM. This probe was successfully used to distinguish the
different levels of NAD(P)H in HepG2 cells under normoxic
and hypoxic conditions. Furthermore, with the help of TCF-
MQ, we found that high levels of NAD(P)H were produced
before cell death after pharmacological doses of natural
antioxidants (resveratrol, curcumin and celastrol) treatment of
HepG2 cells under hypoxic conditions. The excess NAD(P)H
resulted in reductive stress which might induce the cell death.
Scheme 2. TCF-MQ for Fluorescent Detection of NAD(P)H
Fluorescent performance analysis of TCF-MQ.
The spectral properties of the probe with or without NADH
were measured in PBS buffer solution (0.5 % DMSO, 10 mM
PBS, pH = 7.4). As shown in Figure 1a, the probe displayed a
weaker fluorescence peak at 604 nm (λex = 576 nm). As
expected, after the reaction of the probe with NADH, a
significant fluorescence enhancement at 610 nm (λex = 582 nm)
was observed (Quantum Yield = 0.064). Meanwhile, the
fluorescence intensity at 610 nm was enhanced gradually and
reached a plateau within 25 min (Fig. 1b). The effect of pH on
the fluorescent detection of NADH with TCF-MQ was also
investigated, which indicated that TCF-MQ responded to
NADH well under physiological conditions (at 37 °C and pH
=7.4) (Fig. S3).
Results and discussion
Design and Synthesis of TCF-MQ.
2-Dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran
(TCF) as an strong electron-withdrawing group has been widely
used in non-linear optical materials33,34. For TCF-based
fluorophores typically have
a donor-π-acceptor (D-π-A)
structure with long emission wavelengths, many fluorescent
probes have been developed35. Here, we designed and
synthesized a TCF-based fluorescent probe (TCF-MQ) for the
detection of NAD(P)H (Scheme 1). TCF-MQ and all
1
intermediate structures were fully characterized by H NMR,
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of 0-3 μM (Fig. 2a). The regression equation is VFiew=Art8ic3le.2O2nlin+e
DOI: 10.1039/C9SC02020A
238.42 [NADH], with a linear coefficient of 0.9819. The limit
of detection (LOD) was calculated to be as low as 6 nM on the
basis of S/N=3, which indicates that TCF-MQ has excellent
sensitivity to NADH and is more suitable for detecting NADH
in living systems than DCI-MQ (LOD=12 nM)26.
Selective recognition of NAD(P)H by TCF-MQ.
To illustrate the good selectivity of TCF-MQ toward
NAD(P)H, a series of biologically relevant species including
inorganic salts, amino acids and reducing agents were evaluated.
As shown in Figure 3, compared with NADPH and NADH,
inorganic salts (K+, Na+, Ca2+, Mg2+, Zn2+, Fe3+, Fe2+), amino
acids (glycine, lysine, methionine, serine, cysteine,
homocysteine), reducing agents (glutathione, Vitamin C,
dithiothreitol, H2S2, SO32-), NAD+, NADP+ and FADH2 did not
cause obvious changes of the fluorescence intensity indicating
not interfere with the detection of NAD(P)H. In addition, the
reactivity of TCF-MQ toward ROS was also tested. The results
showed that biologically relevant ROS, including O2, H2O2,
NaClO, t-BuOOH and NO, did not trigger any fluorescence
changes in the probe solution (Fig. S5). These results
demonstrate that TCF-MQ can be employed for specific
recognition of NAD(P)H with high selectivity.
Figure 1. (a) Excitation and emission spectra of TCF-MQ (black) and the
product of TCF-MQ with NADH (red). (b) Time-dependent fluorescence
changes of TCF-MQ (10 μM) upon addition of NADH (50 μM).
Next, we investigated the sensitivity of the probe to NADH.
The spectra of the solution of TCF-MQ treated with different
concentrations of NADH (0 to 50 μM) were recorded. Upon
treatment with NADH, the fluorescence intensities at 610 nm
gradually increased with increasing concentrations of NADH
(Fig. S4), and there was a good linearity between the
fluorescence intensities and NADH concentrations in the range
Figure 3. Fluorescence responses of probe TCF-MQ (10 μM) for various
analytes: 1 mM of (K+, Na+, Ca2+, Mg2+, Zn2+, Fe3+, Fe2+, GSH, Gly, Lys,
Met, Ser); 100 μM of (Cys, Hcy, VC, DTT); 5 μM of (H2S2, SO32-); and 50
μM of (NAD+, NADP+, FADH2, NADPH, NADH).
Bioimaging of NAD(P)H in living cells.
After confirming the high sensitivity and selectivity of TCF-
MQ for NAD(P)H, we explored its applications to image
endogenous NAD(P)H in living cells. Before applied to cell
imaging, we first tested the cytotoxicity of this probe by a MTT
assay. The result showed that the TCF-MQ probe exhibited
negligible cytotoxicity when HepG2 cells were treated with 0-
50 μM probe for 18 h (Fig. S6). The resistance to
photobleaching experiments showed that no obvious
fluorescence decrease was observed when cells were exposured
to the laser radiation for 600s (Fig. S7), suggesting the probe is
stable and can be used as a viable probe in living cells.
Figure 2. (a) Fluorescence intensities changes of TCF-MQ (10 μM) upon
addition of different concentrations of NADH (0−3 μM) in PBS (10 mM, pH
7.4, 0.5% DMSO as acosolvent) at 37 oC for 30 min. (b) A linear correlation
between emission intensities and concentrations of NADH. λex = 582 nm,
λem = 610 nm, slit width: 5 nm /5 nm.
Previous studies have shown that hypoxia can enhance
NAD(P)H levels 36,37. Here, we investigated the imaging ability
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of TCF-MQ for NAD(P)H in HepG2 cells under hypoxia (1% detected by confocal microscopy. Under hypoxic conditions, the
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O2) and normoxia (20% O2), respectively. HepG2 cells were HepG2 cells treated with 0-10 μg/mL resDveOrIa: 1tr0o.1l03f9o/rC69SCh02h0a2d0Aa
cultured under hypoxic or normoxic conditions for 12 h, and concentration-dependent increase in fluorescence. However, when
then incubated with 10 μM TCF-MQ for 5-25 min. As shown in the resveratrol concentration reached 20 μg/mL, the fluorescence
Figure 4, the fluorescence of TCF-MQ reached the brightest intensity decreased due to the cell was going to die (Fig. 5a, d and
within 15 min, and the fluorescence intensity under hypoxia g). Additionally, after the HepG2 cells were treated with 10 μg/mL
was higher than that under normoxia, indicating that more resveratrol for 0-9 h, the fluorescence intensity was also enhanced
NAD(P)H was generated in cells under hypoxia. This result in a time-dependent manner (Fig. S8). In contrast, under normoxic
indicates that the probe can be used to monitor the levels of conditions, after the HepG2 cells were exposed to 0-20 μg/mL
NAD(P)H in living cells.
resveratrol for 6 h or 10 μg/mL resveratrol for 0-9 h, the cells
exhibited much lower fluorescence compared with that under the
hypoxic conditions, and there was no enhancement of fluorescence
in the process of cell death (Fig. 5a, d and g, Fig. S8). Curcumin
and celastrol showed similar results to resveratrol. When HepG2
cells were treated with curcumin or celastrol under hypoxic
conditions, the fluorescence intensity of NAD(P)H was increased
in a concentration- and time-dependent manner, but decreased
with cell death (Fig. 5b-i). However, under normoxic conditions,
only intracellular background fluorescence of TCF-MQ was
observed during cell death induced by curcumin and celastrol
(Fig. S9 and 10). These results indicate that reductive stress is
involved in the process of HepG2 cell death induced by
resveratrol, curcumin and celastrol, and the cell death may be
attributed to the reductive stress.
Because previous studies have shown that natural
antioxidants (resveratrol, curcumin and celastrol) can stimulate
the production of ROS in tumor cells cultured in vitro4-6, we
assessed whether oxidative stress was induced as HepG2 cells
were treated with the above three natural antioxidants under
hypoxic conditions. In this experiment, a previously described
H2O2 probe was employed38. HepG2 cells were exposed to
resveratrol (10 μg/mL), curcumin (10 μg/mL) and celastrol (
2
μg/mL for 0-9 h under hypoxic conditions, respectively. Then, the
)
cells were incubated with the H2O2 probe for 15 min. As a result,
there were no obvious increase in H2O2 levels in the three
antioxidant treatment groups compared with the control group (Fig.
S11-13). These results indicate that the three antioxidants do not
cause oxidative stress under hypoxic conditions.
To further confirm the reductive stress induced by natural
antioxidants in HepG2 cells under hypoxic conditions, we
conducted a similar experiment in vivo. Tumor bearing mice
were treated with different concentrations of resveratrol,
curcumin and celastrol via subcutaneous injection at the tumor
site for 10 days. After treatment, the mice received a
subcutaneous administration of the TCF-MQ probe (10 μM)
into the tumor and incubated for 30 min. Then, the fluorescence
images were obtained using an in vivo imaging system. The
fluorescence signal of the probe was obviously increased in a
dose-dependent manner in the resveratrol treatment groups (Fig.
6a and d). Similar to the resveratrol results, the fluorescence
intensity of the probe was also enhanced in the curcumin and
celastrol treatment groups (Fig. 6b, c, e and f), indicating
NAD(P)H levels were elevated during the three natural
antioxidants treatment of liver cancer in mice. These findings
support the in vitro results obtained using the HepG2 cells under
hypoxia showing that reductive stress was induced by natural
Figure 4. Fluorescence imaging of NAD(P)H in living cells. HepG2 cells
were cultured under hypoxic (1% O2) or normoxic (20% O2) conditions for
12 h, and then incubated with 10 μM of the TCF-MQ probe for 5-25 min. (a)
The fluorescence images were obtained using confocal microscopy. (b) The
fluorescence intensity was quantified based on the results of the relative
fluorescence intensity of per cell in the scanned area. The scale bar in all
fluorescence images of cells is 20 μm.
Revealing reductive stress induced by natural antioxidants.
Next, we used the TCF-MQ probe to observe the NAD(P)H
levels in the process of liver cancer cell death induced by
natural antioxidants (resveratrol, curcumin and celastrol).
HepG2 cells were exposed to different concentrations of resveratrol,
curcumin and celastrol for different time under hypoxic and
normoxic conditions, respectively. After treatment, the cells were
loaded with 10 μM TCF-MQ for 20 min, then, the fluorescence was
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DOI: 10.1039/C9SC02020A
Figure 5. Detection of NAD(P)H levels induced by natural antioxidants in living cells. (a) HepG2 cells were exposed to 0-20 μg/mL resveratrol for 6 h
under hypoxic conditions (1%O2) and normoxic conditions (20%O2), then the cells were incubated with 10 μM of the TCF-MQ probe for 20 min before the
fluorescence images were obtained using confocal microscopy. (b) HepG2 cells were exposed to 0-20 μg/mL curcumin for 6 h under hypoxic conditions
(1%O2) and normoxic conditions (20%O2), then the cells were treated using the method described in (a). (c) HepG2 cells were exposed to 0-4 μg/mL
celastrol for 6 h under hypoxic conditions (1%O2) and normoxic conditions (20%O2), then the cells were treated using the method described in (a). (d-f) The
fluorescence intensity of Figure a-c were quantified based on the results of the relative fluorescence intensity of per cell in the scanned area. (g) HepG2 cells
were exposed to 0-20 μg/mL resveratrol for 6 h under hypoxic conditions (1%O2) and normoxic conditions (20%O2), then the cells were collected and
stained by trypan blue. (h) HepG2 cells were exposed to 0-20 μg/mL curcumin for 6 h under hypoxic conditions (1%O2) and normoxic conditions (20%O2),
then the cells were collected and stained by trypan blue. (i) HepG2 cells were exposed to 0-4 μg/mL celastrol for 6 h under hypoxic conditions (1%O2) and
normoxic conditions (20%O2), then the cells were collected and stained by trypan blue. (*p<0.05, **p<0.01, t test). The scale bar in all fluorescence images
of cells is 20 μm.
antioxidants treatment. The tumor volume measurement
experiments showed that resveratrol could significantly inhibit
tumor growth in a dose- and time-dependent manner (Fig. 6g).
Conclusions
Curcumin and celastrol could also suppress the tumor growth in
In summary, we developed
a
novel small-molecule
mice (Fig. 6h and i), indicating the reductive stress induced by
three natural antioxidants can inhibit the growth of liver cancer
tumor.
fluorescent probe, TCF-MQ, for monitoring of NAD(P)H and
revealing the reductive stress. TCF-MQ can rapidly respond to
NAD(P)H with good selectivity and exhibited very high
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Figure 6. Detection of NAD(P)H levels induced by natural antioxidants in vivo. (a) Tumor-bearing mice were treated with 0-30μg/mL resveratrol for 10
days, and then, 10 μM TCF-MQ probe was subcutaneously injected into the tumor for 30 min before the fluorescence was analyzed using an in vivo imaging
system. (b) Tumor-bearing mice were treated with 0-30μg/mL curcumin for 10 days, then the TCF-MQ probe was added using the method described in (a).
(c) Tumor-bearing mice were treated with 0-4 μg/mL celastrol for 10 days, then the TCF-MQ probe was added using the method described in (a). (d) The
quantitative fluorescence intensity for (a). (e) The quantitative fluorescence intensity for (b). (f) The quantitative fluorescence intensity for (c). (g) Tumor-
bearing mice were treated with 0-30μg/mL resveratrol for different days, the tumor volumes were measured using digital calipers. (h) Tumor-bearing mice
were treated with 0-30μg/mL curcumin for different days, the tumor volumes were measured using digital calipers. (i) Tumor-bearing mice were treated with
0-30μg/mL 0-4 μg/mL celastrol for different days, the tumor volumes were measured using digital calipers. (*p<0.05, **p<0.01, ***p<0.001, t test).
sensitivity to NAD(P)H with a low detection limit of 6 nM. The resveratrol, curcumin and celastrol and provide a new research
probe was successfully used to image endogenous NAD(P)H in area for natural antioxidants
living cells and to distinguish the different levels of NAD(P)H
.
in HepG2 cells under normoxic conditions and hypoxic
conditions. Moreover, with the help of TCF-MQ, we found that
Experimental
pharmacological doses of three natural antioxidants (resveratrol,
Materials and methods
.
3-Hydroxy-3-methyl-2-butanone,
curcumin and celastrol) treatment of HepG2 cells under
hypoxic conditions, high levels of NAD(P)H were produced
before cell death. The excess NAD(P)H resulted in reductive
stress instead of oxidative stress. In contrast, under normoxic
malononitrile, methyl trifluoromethanesulphonate and 3-
quinolinecarboxaldehyde were purchased from Sun Chemical
Technology (Shanghai, China). β-NADPH and β-NADH were
obtained from Aladdin (Shanghai, China). Piperidine, sodium
ethoxide, anhydrous ethanol, chloroform and acetonitrile were
from Sinopharm Chemical Reagent (Shanghai, China).
Resveratrol, Curcumin and Celastrol were from Dalin Meilun
Biotecnology (Dalian, China). 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphnyl-2H-tetrazolium bromide (MTT) was from Sigma-
Aldrich (St. Louis, MO, USA). 1H NMR and 13C NMR
spectrum were taken on a nuclear magnetic resonance
spectrometer (Advance 400 MHz, Bruker, Switzerland). High-
resolution mass spectralanalyses (HRMS) were recorded on a
conditions
in the process of cell death induced by resveratrol, curcumin and
celastrol Thus, we speculate that natural antioxidants might induce
, there were no obvious increase in NAD(P)H contents
.
tumor cell death via reductive stress under tumor hypoxic
microenvironment, whereas under normoxic environment, natural
antioxidants induced cell death through oxidative stress based on the
previous reports4-6. The mechanism of reductive stress inducing
tumor cell death under hypoxic conditions is undergoing further
research. Our findings reveal a new anti-cancer mechanism of
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High Resolution Quadrupole-time of flight mass spectrometer prepared from a solution of KO2 in DMSO. For spectral
View Article Online
DOI: 10.1039/C9SC02020A
(Bruker, Germany). Fluorescence spectra were carried out on a measurements, the stock solution of the TCF-MQ probe was
fluorescence spectrometer (Edinburgh Instruments, U.K.). The diluted with PBS buffer to give a final concentration of 10 μM.
fluorescence imaging was obtained with a confocal (LSM 880, NAD(P)H and various analytes were added to the solution of
Zeiss, Germany).
TCF-MQ (10 μM) in PBS buffer (10 mM, pH = 7.4, containing
0.5% DMSO). The fluorescence emission spectra were
recorded in a range from 592–800 nm (λex = 582 nm, λem =
Synthesis of probe TCF-MQ.
Synthesis of compound 1. 3-hydroxy-3-methyl-2-butanone (1 610 nm, slit width: 5 nm/5 nm).
mL, 9.5 mmol) and malononitrile (1.3 g, 20 mmol) were added Cell culture. The cell culture refered to our previous paper39.
into 8 mL anhydrous ethanol respectively to form a solution, Briefly, HepG2 cells were cultured in DMEM (high glucose)
then, sodium ethoxide (0.1 g, 1.5 mmol) was added, and the supplemented with 10% FBS and 1% antibiotics (100 U/mL
solution was stirred at room temperature for 1.5 h, and then penicillin and 100 mg/mL streptomycin) at 37 °C in a
refluxed at 60 oC for another 1 h. After cooling to room humidified atmosphere of 95% air (20% O2) and 5% CO2. For
temperature, the precipitate was filtered to give compound 1 simulating the tumor hypoxic microenvironment, HepG2 cells
1
(1.1 g, yield: 60%). H NMR (Chloroform-d, 400MHz): δ= were cultured in a mixture containing 1% O2, 94% N2 and 5%
2.37 (s, 3H), 1.63 (s, 6H); 13C NMR (Chloroform-d, 101MHz): CO2 using a low oxygen incubator (Bugbox M, Ruskinn,
δ=182.69, 175.25, 111.06, 110.44, 109.00, 104.80, 99.82, 58.44, England).
24.37, 14.23. HRMS: (ESI, m/z) Calcd for C11H9N3O [MH]:
198.0661, found: 198.0694.
Fluorescence imaging in living cells. The fluorescence of
Synthesis of compound 2. Compound 1 (160 mg, 0.8 mmol) NAD(P)H and H2O2 was detected by confocal microscopy.
and 3-quinolinecarboxaldehyde(151 mg, 0.96 mmol) were Briefly, HepG2 cells (5 × 104) were seeded on glass-bottom
dissolved in 15 mL anhydrous ethanol, two drops piperidine culture dishes (15 mm) for 24 h. Then, the cells were exposed
and acetic acid were added and the solution was refluxed for 2 to different concentrations of three natural antioxidants for
h. After cooling to room temperature, the precipitate was different time under hypoxic and normoxic conditions,
1
filtered to obtain compound 2 (190 mg, yield: 70%). H NMR respectively. After treatment, the cells cultured under hypoxia
(DMSO-d6, 400MHz): δ=9.41 (d, J = 2.2 Hz, 1H), 8.97 (d, J = and normoxia were simultaneously incubated with 10 μM (1mL)
2.2 Hz, 1H), 8.17–8.02 (m, 3H), 7.88 (t, J = 8.0 Hz, 1H), 7.71 (t, NAD(P)H probe for 20 min or 10 μM (1mL) H2O2 probe for 15
J = 7.5 Hz, 1H), 7.54 (d, J = 16.6 Hz, 1H), 1.86 (s, 6H); 13C min in FBS-free DMEM medium at 37 °C, and then, cells were
NMR (DMSO-d6, 101MHz): δ=177.55, 174.96, 150.90, 148.84, washed three times with PBS buffer and imaged immediately
144.48, 137.41, 132.15, 129.73, 129.37, 128.15, 128.08, 127.69, using a confocal microscope. TCF-MQ probe was excited at
117.49, 113.10, 112.26, 111.20, 100.91, 100.07, 55.37, 25.56. 582 nm and the fluorescence emission range was set from 600-
HRMS: (ESI, m/z) Calcd for C21H14N4O [M+H]+: 339.1240, 650 nm. H2O2 probe was excited at 532 nm and the
found: 339.1208.
fluorescence emission range was set from 600-700 nm.
Synthesis of TCF-MQ. A mixture of compound 2 (60 mg.
0.18 mmol) and methyl trifluoromethanesulfonate (0.1 mL, Tumor model preparation and fluorescence imaging. All
0.92mmol) in 5 mL trichloromethane was stirred at room animal experiments were performed according to the Principles
temperature for 24 h under nitrogen atmosphere. Ether was of Laboratory Animal Care (People's Republic of China) and
added after the solvent was removed in vacuo, and then the the Guidelines of the Animal Investigation Committee, and
precipitate was filtered to give TCF-MQ (68 mg, yield: 75%). approved by the local Animal Care and Use Committee.
1H NMR (DMSO-d6, 400MHz): δ= 10.05 (s, 1H), 9.72 (s, 1H), Kunmin mice were purchased from the Qingdao Daren
8.56-8.54 (d, J = 8.0 Hz, 1H), 8.49-8.47 (d, J = 8.0 Hz, 1H), Fucheng Animal Co., Ltd. Hepatocellular carcinoma H22 cells
8.36-8.34 (q, J = 8.0 Hz, 1H), 8.13–8.09 (q, J = 8.0 Hz,1H), (1×107) derived from mice were injected into the abdominal
8.07-8.03 (d, J = 16.0 Hz, 1H), 7.66-7.62 (d, J = 16.0 Hz, 1H), cavity of mice, the ascites were formed after 5-7 days. After
4.67 (s, 3H), 1.87 (s, 6H); 13C NMR (DMSO-d6, 101MHz): three passages, the ascites were used. We selected 8-week-old
δ=177.27, 173.43, 150.93, 146.26, 140.07, 138.57, 137.12, mice and injected H22 cells subcutaneously into the axillary
131.69, 131.21, 129.19, 128.82, 120.28, 119.91, 112.86, 112.05, lateral of the left forelimb at a density of 1×107 cells/mL with
110.81, 103.48, 100.05, 100.00, 56.45, 46.18, 25.50. HRMS: 100 μL per mice. After 7 days, these mice were given different
(ESI, m/z) Calcd for C23H17F3N4O4S [MTfO]+: 353.1396, concentrations of natural antioxidants through subcutaneous
found: 353.1385.
injection at the tumor site for different time. After treatment,
the mice were subcutaneously injected with the TCF-MQ probe
In vitro detection of NAD(P)H. The stock solution of TCF- and incubated for 30 min. Then, the fluorescence images were
MQ (210-3 M) was prepared in DMSO. Stock solutions of obtained using an in vivo imaging system (IVIS) with 582 nm
NAD(P)H and various interferential reagents (Na+, K+, Ca2+, excitation and 600-650 nm collection for NAD(P)H. In addition,
Mg2+, Zn2+, Fe2+, Fe3+, glycine, lysine, serine, methionine, the tumor volume was measured by digital calipers and
cysteine, homocysteine, glutathione, dithiothreitol, Vitamin C, calculated as: volume = 0.5 × (length×width2)40.
and H2O2, NaClO, t-BuOOH, NO) were prepared in double-
distilled water, but superoxide anion free radical (O2) was
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19 H. Komatsu, Y. Shindo, K. Oka, J. P. Hill, K. Ariga, Angew.
Conflicts of interest
There are no conflicts to declare.
View Article Online
DOI: 10.1039/C9SC02020A
Chem. Int. Ed. Engl. 2014, 53, 3993-3995.
20 V. A. Aleshin, A. V. Artiukhov, H. Oppermann, A. V.
Kazantsev, N. V. Lukashev, V. I. Bunik, Cells. 2015, 4, 427-
451.
21 L. Yuan, W. Y. Lin, K. B. Zheng, L. W. He, W. M. Huang,
Chem. Soc. Rev. 2013, 42, 622–661.
22 J. Yin, Y. Kwon, D. Kim, D. Lee, G. Kim, Y. Hu, J. H. Ryu,
J. J. Yoon, Am. Chem. Soc. 2014, 136, 5351-5358.
23 X. Ao, S. A. Bright, N. C. Taylor, R. B. P Elmes, Org.
Biomol. Chem. 2017, 15, 6104-6108.
24 A. Tirla, P. Rivera-Fuentes, Angew. Chem. Int. Ed. Engl.
2016, 55, 14709-14712.
Acknowledgements
This work was supported by National Natural Science
Foundation of China (21535004, 21575081, 21775091 and
91753111), the Key Research and Development Program of
Shandong Province (2018YFJH0502), the Shandong Provincial
Natural Science Foundation of China (ZR2014JL055) and the
key research and development plan of Yantai City
(2019XDHZ098).
25 A. Tirla, P. Rivera-Fuentes, Chimia (Aarau). 2018, 72, 241-
244.
26 C. A. Roeschlaub, N. L. Maidwell, M. R. Rezai, P. G.
Sammes, Chem. Commun. 1999, 1637-1638.
27 C. Tu, R. Nagao, A. Y. Louie, Angew. Chem. Int. Ed. 2009,
48, 6547-6551.
28 S. O. Jung, J. Y. Ahn, S. Kim, Tetrahedron. Letters. 2010, 51,
3775-3778.
Notes and references
1
D. Sinha, N. Sarkar, J. Biswas, A. Bishayee, Semin. Cancer.
Biol. 2016, 40-41, 209-232.
2
J. Y. Wang, X. Wang, X. J. Wang, B. Z. Zheng, Y. Wang, X.
Wang, B. Liang, Eur. Rev. Med. Pharmacol. Sci. 2018, 22,
7492-7499.
3
4
Shrivastava S, Jeengar MK, Reddy VS, Reddy GB, Naidu
VG, Exp. Mol. Pathol. 2015, 98, 313-327.
D. J. Colin, E. Limagne, K. Ragot, G. Lizard, F. Ghiringhelli,
É. Solary, B. Chauffert, N. Latruffe, D. Delmas, Cell. Death.
Dis. 2014, 5, e1533.
F. Song, L. Zhang, H. X. Yu, R. R. Lu, J. D. Bao, C. Tan, Z.
Sun, Food. Chem. 2012, 132, 43-50.
J. H. Kim, J. O. Lee, S. K. Lee, N. Kim, G. Y. You, J. W.
Moon, J. Sha, S. J. Kim, S. H. Park, H. S. Kim, Cell. Signal.
2013, 25, 805-813.
29 H. Komatsu, Y. Shindo, K. Oka, I. P. Hill, K. Ariga, Angew.
Chem. Int. Ed. 2014, 53, 3993-3995.
30 M. A. Fomin, R. I. Dmitrev, J. Jenkins, D. B. Papkovsky, D.
Heindl, B. Konig, ACS. Sens. 2016, 1, 702-709.
31 L. Wang, J. Zhang, B. Kim, J. Peng, S. N. Berry, Y. Ni, D.
Su, J. Lee, L. Yuan, Y. T. Chang, J. Am. Chem. Soc. 2016,
138, 10394-10397.
32 Y. Zhao, K. Wei, F Kong, X. Gao, K. Xu, B. Tang, Anal.
Chem. 2018, 91, 1368-1374.
33 C. Zhang, L. R. Dalton, M-C. Oh, H. Zhang, W. H. Steier,
Chem. Mater. 2001, 13, 3043-3050.
34 W. Bentoumi, J. C. Mulatier, P. A. Bouit, O. Maury, A.
Barsella, J. P. Vola, E. Chastaing, L. Divay, F. Soyer, P. Le
Barny, Y. Bretonnière, C. Andraud, Chemistry. 2014, 20,
8909-8913.
5
6
7
8
9
L. J. Yan, J. Diabetes. Res. 2014, 2014, 137919.
E. B. Rankin, A. J. Giaccia, Science. 2016, 352, 175-180.
N. C. Denko, Nat. Rev. Cancer. 2008, 8, 705-713.
10 X. X. Xiong, X. Y. Qiu, D. X. Hu, X. Q. Chen, Mol.
Pharmacol. 2017, 92, 246-255.
11 X. X. Fan, H. D. Pan, Y. Li, R. J. Guo, E. L. Leung, L. Liu,
Pharmacol. Ther. 2018, 191, 148-161.
12 M. Inoue, R. Nakashima, M. Enomoto, Y. Koike, X. Zhao, K.
Yip, S. H. Huang, J. N. Waldron, M. Ikura, F. F. Liu, S. V.
Bratman, Nat. Commun. 2018, 9, 5116.
13 B. Petrova, K. Liu, C. Tian, M. Kitaoka, E. Freinkman, J.
Yang, T. L. Orr-Weaver, Proc. Natl. Acad. Sci. U S A. 2018,
115, E7978-E7986.
35 A. C. Sedgwick, H. H. Han, J. E. Gardiner, S. D. Bull, X. P.
He, T. D. James, Chem. Commun (Camb). 2017, 53, 12822-
12825.
36 L. B. Jiang, L. Cao, Y. Q. Ma, Q. Chen, Y. Liang, F. L. Yuan,
X. L. Li, J. Dong, N. Chen, Osteoarthritis. Cartilage. 2018,
26, 138-148.
37 O. Polesskaya, A. Sun, G. Salahura, J. N. Silva, S. Dewhurst,
K. Kasischke, J. Vis. Exp. 2012, pii: 3466.
14 N. S. Rajasekaran, P. Connell, E. S. Christians, L. J. Yan, R.
P. Taylor, A. Orosz, X. Q. Zhang, T. J. Stevenson, R. M.
Peshock, J. A. Leopold, W. H. Barry, J. Loscalzo, S. J.
Odelberg, I. J. Benjamin, Cell. 2007, 130, 427-439.
15 H. Zhang, P. Limphong, J. Pieper, Q. Liu, C. K. Rodesch, E.
Christians, I. J. Benjamin, FASEB J. 2012, 26, 1442-1451.
16 D. E. Handy, J. Loscalzo, Free. Radic. Biol. Med. 2017, 109,
114-124.
38 X. Liu, B. Hu, R. Cheng, F. Kong, X. Pan, K. Xu, B. Tang,
Chem. Commun (Camb). 2016, 52, 6693-6696.
39 X. H. Pan, X. X. Song, C. Wang, T. T. Cheng, D. R. Luan, K.
H. Xu, B. Tang, Theranostics. 2019, 9, 1794-1808.
40 W. S. Henry, T. Laszewski, T. Tsang, F. Beca, A. H. Beck, S.
S. McAllister, A. Toker, Cancer. Res. 2017, 77, 790-801.
17 S. Dimmeler, A. M. Zeiher, Cell. 2007, 130, 401-402.
18 A. C. Brewer, S. B. Mustafi, T. V. Murray, N. S.
Rajasekaran, I. J. Benjamin, Antioxid. Redox. Signal. 2013,
18, 1114-1127.
8 | J. Name., 2012, 00, 1-3
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Chemical Science
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DOI: 10.1039/C9SC02020A
An ultrasensitive fluorescent probe for monitoring NAD(P)H and revealing reductive stress
induced by natural antioxidants in HepG2 cells under hypoxia.