A fluorescent probe for simultaneously sensing NTR and hNQO1 and distinguishing cancer cells

Article abstract of DOI:10.1039/c9tb01581g

Identifying cancer at the cellular level during an early stage offers the hope of greatly improved outcomes for cancer patients. As potential cancer biomarkers, nitroreductase (NTR) and human quinine oxidoreductase 1 (hNQO1) are overexpressed in many type

Full text of DOI:10.1039/c9tb01581g

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A fluorescent probe for simultaneously sensing
NTR and hNQO1 and distinguishing cancer cells†
Cite this: J. Mater. Chem. B, 2019,
7, 6822
Fanpeng Kong,‡ Ying Li,‡ Chao Yang, Xiao Li, Junlin Wu, Xiaojun Liu, Xiaonan Gao,
Kehua Xu* and Bo Tang
*
Identifying cancer at the cellular level during an early stage offers the hope of greatly improved
outcomes for cancer patients. As potential cancer biomarkers, nitroreductase (NTR) and human quinine
oxidoreductase 1 (hNQO1) are overexpressed in many type of cancer cells. Simultaneous detection of
these two biomarkers would benefit diagnostic precision in related cancers without yielding false
positive results. Herein, based on a dye generated in situ strategy, a dual-enzyme-responsive probe, CNN,
was rationally designed and synthesized by installing p-nitrobenzene and trimethyl-locked quinone
propionic acid groups, which are specific for NTR and hNQO1, respectively, into a single fluorophore. This
probe is only activated in the presence of both NTR and hNQO1 and produces a large fluorescence
response, enabling the detection of both endogenous NTR and hNQO1 activity in living cells. The imaging
results indicate that the CNN probe differentiates cancer cells (HeLa, MDA-MB-231 and HepG2 cells) from
normal liver HL-7702 cells owing to the existence of relatively high endogenous levels of both biomarkers
in these cancer cells.
Received 29th July 2019,
Accepted 29th September 2019
DOI: 10.1039/c9tb01581g
rsc.li/materials-b
non-small cell lung cancer and is associated with aggressive
tumors and poor patient outcomes.6 Overexpressed thioredoxin
Introduction
Cancer is a substantial public health problem and the second reductase (TrxR) is expressed at high levels in a wide variety
leading cause of death worldwide.1,2 Most cancer deaths are of human tumors including lung, pancreas, colon, gastric
caused by the spread of cancer cells from primary tumor cells to and breast cancer.7 Cytochrome P450 1A (CYP 1A) is highly
distal organs (metastasis). Therefore, the survival of cancer expressed in a wide range of tumor types and may greatly
patients is strongly associated with the tumor stage at the time contribute to the development and progression of cancer.8
of diagnosis. Clinical identification of tumor biomarkers holds Detection of tumor-related overexpression of redox enzymes
great potential for rapid prediction of cancer diagnosis and offers provides new tools for discriminating a wide range of cancer
the hope of greatly improved outcomes for cancer patients.3 cells both in vitro and in vivo. Fluorescence-guided diagnostics
Among numerous tumor markers, overexpressed proteases are is one of the most powerful real-time techniques for in situ tumor
promising candidate enzymes to leverage both diagnostically and detection9 due to its high sensitivity, low cost, portability, and real-
therapeutically as they play critical roles in cancer progression.4
time capabilities.10–12 Multiple fluorescent probes for detecting
Redox enzymes regulate redox homeostasis in living systems, tumor specific redox enzymes and other protease overexpression
and their abnormal regulation is implicated in a variety of in complex biological specimens have been reported.13–25
cancers. For example, the activity of methionine sulfoxide
Nitroreductase (NTR) is an indicator of highly aggressive disease
reductases is known to be a crucial biomarker for human breast in various hypoxic tumor cells,26,27 and overexpression of NTR
cancers.5 Tyrosine oxidase is implicated in melanoma cancer. during hypoxia plays an important role in malignant tumor progres-
Cyclooxygenase
2
(COX-2) is frequently overexpressed in sion and angiogenesis. Human NAD(P)H: quinone oxi-doreductase
isozyme I (hNQO1) is markedly upregulated in many types of cancer
cells and solid tumors.28 Importantly, hNQO1 overexpression has
College of Chemistry, Chemical Engineering and Materials Science, Institute of
Biomedical Sciences, Collaborative Innovation Center of Functionalized Probes for
Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and
Nano Probes, Ministry of Education, Shandong normal University, Jinan 250014,
P. R. China. E-mail: tangb@sdnu.edu.cn, xukehua@sdnu.edu.cn
been directly linked to both tumor genesis and poor patient
survival.29 Therefore, early and accurate NTR and hNQO1 detection
is likely important for cancer diagnosis. Many fluorescence probes
have been synthesized for the detection and imaging of NTR30–36
and hNQO137–42 in cancer cells. However, detecting a single tumor
related overexpressed protease may yield false positive results,
restricting identification of cancer.
† Electronic supplementary information (ESI) available: Experimental proce-
dures, synthesis of the probe, 1H NMR, 13C NMR and HRMS data and MTT
assay. See DOI: 10.1039/c9tb01581g
‡ These authors contributed equally to this work.
6822 | J. Mater. Chem. B, 2019, 7, 6822--6827
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Simultaneous detection of multiple tumor markers brings In contrast, the p-nitrobenzene group was reduced to the amino-
new opportunities for improving the accuracy of early cancer benzene group by NTR in the presence of NADH, followed
detection compared to the single-marker assay.43–45 Recently, by a 1,6-rearrangement elimination reaction, resulting in the
various platforms using nanomaterials for the detection of multiple formation of a coumarin derivative (compound B). However, the
targets have been developed,46–50 but relatively few molecular fluorescence of compound B was very low because the Q3PA
probes have been developed that detect multiple tumor markers group is an effective photo-induced electron transfer (PeT)
simultaneously.51 Herein, we report a dual site fluorescence probe, quencher of fluorescence due to its electron deficient nature.
CNN, to which a 4-nitrobenzene moiety and trimethyl-locked Treatment of CNN with NTR and hNQO1 together resulted in
quinone propionic acid (Q3PA) were introduced as reaction units, strong fluorescence enhancement due to formation of 7-hydroxy-
as well as a fluorescence quencher. In the presence of NTR and (2-imino)coumarin (quantum yield, F = 0.16) (Fig. S1, ESI†).
hNQO1, CNN exhibited a dramatic off–on fluorescence response, The sensing mechanism of CNN toward NTR and hNQO1 was
while other potential interfering species, including inorganic salts, validated by using HRMS (Fig. S2, ESI†). The synthesis route for
amino acids, thiols, reactive oxygen species (ROS) and glucose, the CNN probe is shown in Scheme 1. The structures of all
elicited no significant fluorescence. Cellular imaging experi- intermediates and the final product were confirmed by nuclear
ments clearly indicated that CNN differentiates cancer cells magnetic resonance (NMR) and high resolution mass spectro-
from normal cells owing to the existence of relatively high metry (HRMS) (see the ESI†).
endogenous levels of both biomarkers.
The fluorescence responses of the CNN probe to different
concentrations of NTR and hNQO1 were investigated. As shown
in Fig. 1a, CNN was initially non-fluorescent, and with the
addition of NADH (50 mM) and hNQO1 (0.4 mg mLÀ1) almost
no fluorescence increase was observed. However, subsequent
Results and discussion
The CNN probe design is based on in situ formation of blue additions of NTR resulted in evident increases in fluorescence
emitting 7-hydroxy (2-imino)coumarin52–54 generated from a intensity. Furthermore, a good linear relationship exists between
caged bifunctional precursor through effective domino reactions the fluorescence intensity at 454 nm and concentrations of
triggered by NTR and hNQO1 (Scheme 1a). The p-nitrobenzene NTR in the 0–2.0 mg mLÀ1 range (Fig. 1b), with R2 = 0.992. The
group and trimethyl-locked quinone propionic acid (Q3PA) were detection limit (k = 3) of the probe for NTR was determined as
utilized, which are specific for NTR and hNQO1, respectively. In 10 ng mLÀ1. Emission spectra of CNN were also recorded in
the absence of NTR and hNQO1, the fluorescence of the CNN PBS in the presence of NADH (50 mM) and NTR (2 mg mLÀ1). As
probe was quenched by the electron-withdrawing effect of the 4- shown in Fig. 1c, a slight increase in fluorescence was observed,
nitrobenzene moiety and Q3PA due to PET inhibition. In the and subsequent additions of hNQO1 resulted in a much larger
presence of hNQO1 only, the Q3PA group of the CNN probe was increase in fluorescence intensity. A linear functional relation-
removed, releasing compound A, which still showed no fluores- ship (R2 = 0.993) was obtained between the fluorescence
cence due to linking the 4-nitrobenzene quencher group.
Fig. 1 (a) Fluorescence spectra of CNN (5 mM) after adding different
concentrations of NTR (0–2.4 mg mLÀ1) in the presence of hNQO1
(0.4 mg mLÀ1) and NADH (50 mM). (b) The linear relationship between the
fluorescence intensity and the concentration of NTR. (c) Fluorescence
spectra of CNN (5 mM) after adding different concentrations of hNQO1
(0–0.5 mg mLÀ1) in the presence of NTR (2.0 mg mLÀ1) and NADH (50 mM).
Scheme 1 (a) Potential reaction mechanism of CNN for NTR and hNQO1
detection and (b) synthesis of the probe CNN.
(d) The linear relationship between the fluorescence intensity and the
concentration of hNQO1.
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Fig. 2 (a) Fluorescence responses of CNN (5 mM) upon treatment with
various biologically-relevant species. (b) Fluorescence responses of CNN
(5 mM) against different enzymes.
Fig. 3 (a) Confocal fluorescence microscope images for endogenous
NTR and hNQO1 detection in different cell lines using CNN (10 mM).
(b) Relative fluorescence intensity of the images from (a).
intensity at 454 nm and hNQO1 concentrations (0–0.4 mg mLÀ1
)
with a detection limit as low as 2 ng mLÀ1 based on the 3s/k
principle. These results indicate that the CNN probe requires
both NTR and hNQO1 for a fluorescence response.
The specificity of the CNN probe was evaluated for NTR and
hNQO1 over other potential interfering species, including inorganic
salts, amino acids, thiols, ROS, glucose and two different kinds of
biologically-relevant species (Fig. S3, ESI†). In addition, the selec-
tivity of the probe to carboxylesterase 1 (CES1), carboxylesterase 2
(CES2), glutathione reductase (GR) and P450 cytochrome reductase
in the presence of hNQO1 was also examined. As illustrated in Fig. 2,
addition of other species exhibited a negligible fluorescence
response, with only combined NTR and hNQO1 inducing remark-
able fluorescence enhancement. All data revealed that CNN exerts
excellent specificity under physiological conditions. Furthermore, to
evaluate the compatibility of CNN with biological systems, the
cytotoxicity of the probe was evaluated in HeLa and HepG2 cells
using a standard MTT assay (Fig. S4, ESI†). The cell viability
remained over 80% after incubation with even high concentrations
of the probe (20 mM), indicating good biocompatibility of the
probe with cells. The photostability of the fluorescent product
CNN was also evaluated. After illumination at 405 nm by a 150 W
xenon lamp for 10 min, the fluorescence intensity of CNN buffer
solution remained at 90% (Fig. S5, ESI†), indicating good
photostability of the dye under the detection conditions.
Encouraged by the results described above, we next examined
the potential use of CNN for visualizing NTR and hNQO1 in
living cells to investigate whether CNN could be employed to
distinguish different types of cancer cells. Given the elevated
levels of both NTR and hNQO1 in some cancer cells, three types
of solid tumor cells, HeLa, MDA-MB-231 and HepG2 cells, and
normal liver HL-7702 cells were selected for evaluation. To
simulate the solid tumor hypoxic microenvironment, HeLa,
MDA-MB-231 and HepG2 cells were incubated under hypoxic
Fig. 4 (a) Confocal fluorescence microscope images for endogenous NTR and
hNQO1 detection in HepG2 cells and NCI-H446 cells using CNN (10 mM) under
different conditions. (b) Relative fluorescence intensity of the images from (a).
To further confirm activation of CNN in cancer cells in response
to both NTR and hNQO1, we added dicoumarol, an NTR and
hNQO1 inhibitor, or incubated HepG2 cells with the CNN probe in
aerobic conditions, which causes low expression of NTR. As shown
in Fig. 4a, HepG2 cells incubated with the CNN probe under
hypoxic conditions yielded bright fluorescence. In contrast, in
the inhibitor-treated group and the group incubated in aerobic
conditions, HepG2 cells showed very weak fluorescence with an
observed mean fluorescence intensity only a third of that in the
group without inhibitors (Fig. 4b). In addition, NCI-H446 cells
(a small-cell lung carcinoma known to be devoid of hNQO1
activity)55 were also used for evaluating the role of hNQO1 in the
probe activation. As shown in Fig. 4, even in hypoxic conditions,
the probe cannot be activated in NCI-H446 cells due to the lack of
hNQO1. These results clearly demonstrated the dual NTR and
hNQO1 requirement for CNN function.
conditions (1% pO ) for 4 h compared to the control group
2
(HL-7702 cells in normoxic conditions 20% pO ). Excitingly, all
three cancer cell lines incubated with the CNN probe showed
strong fluorescence in the green channel, whereas almost no
2
Experimental
General chemicals and instruments
intracellular fluorescence was observed in the normal cell line 2,4-Dihydroxybenzaldehyde, imidazole, tert-butyldimethylsilyl
(Fig. 3a). The mean fluorescence intensity in the cancer cell lines chloride, dichloromethane, absolute ethanol, silver oxide, tetra-
was consistently more than 3 times higher than that in the normal hydrofuran, 1-ethyl-(3-dimethylaminopropyl)carbonyldiimide hydro-
cell line (Fig. 3b), indicating that NTR and hNQO1 have the chloride and 4-nitrobenzyl bromide were purchased from Energy
potential to be ubiquitous tumor markers using CNN as a highly Chemical. Malononitrile was purchased from Sun Chemical Tech-
effective probe to differentiate cancer cells from normal cells.
nology Co., Ltd (Shanghai, China). Anhydrous magnesium sulfate,
6824 | J. Mater. Chem. B, 2019, 7, 6822--6827
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anhydrous ether, piperidine, and tetrahydrofuran were purchased which yielded a white solid (2.5 g, 81%). The 1H NMR properties
from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). and findings are as follows: (400 MHz, CDCl3) d 10.18 (s, 1H), 8.07
b-NADH was purchased from Aladdin Reagent Co., Ltd (Shanghai, (d, J = 8.6 Hz, 2H), 7.58 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 8.6 Hz, 2H),
China). b-NADPH was purchased from Aladdin Reagent Co., Ltd 6.32 (d, J = 8.5 Hz, 1H), 6.19 (d, J = 1.8 Hz, 1H), 5.04 (s, 2H), 0.75
(Shanghai, China). NTR from Escherichia coli was purchased from (s, 9H), À0.00 (s, 6H). 13C NMR (101 MHz, CDCl3) d 187.8, 162.9,
Sigma Chemical Company. hNQO1 from Escherichia coli was 161.8, 147.8, 143.3, 131.0, 127.5, 124.0, 119.6, 113.4, 104.6, 77.3,
purchased from ProSpec-Tany TechnoGene Ltd. Recombinant 77.0, 76.7, 69.0, 25.5, À4.3. HRMS, calculated for C20H25NO5Si
Human CES1 is produced by our mammalian expression system [M + H]+ m/z 388.1574, found 388.1567.
and purchased from Novoprotein Scientific Inc. Human CES2
Synthesis of compound 3. Compound 2 (466 mg, 1.2 mmol)
is expressed by human HEK293 cells and purchased from and malononitrile (85 mg, 1.26 mmol) were dissolved in
Sino Biological. Glutathione reductase was purchased from absolute ethanol followed by addition of a catalytic amount of
Solarbio. NADPH-P450 cytochrome reductase was purchased piperidine and stirred at room temperature for 1 hour. The
from ProSpec-Tany TechnoGene Ltd. Dicoumarol was purchased reaction progress was monitored by TLC. After the reaction was
from Medchem Express (MCE). All chemicals and solvents used complete, the solvent was removed under reduced pressure.
for synthesis were purchased from commercial suppliers and The residue was dissolved in tetrahydrofuran (15 mL), and tetra-
applied directly in the experiments without further purification. butylammonium fluoride (1.4 mL) was subsequently added and
1H NMR and 13C NMR spectra were recorded on a Bruker Advance stirred at room temperature for 5 min. The reaction was moni-
400 MHz spectrometer at room temperature. UV-vis absorption tored by TLC. After the reaction was complete, the solvent was
spectra were measured on a UV-visible spectrophotometer removed under reduced pressure. Dichloromethane was added,
(UV-1700 Pharmaspec, Shimadzu, Japan). All pH measurements washed with deionized water, and then washed with 1.0 M HCl
were performed with a pH-3c digital pH-meter (Shanghai LeiCi and brine. The solvent was dried over anhydrous sodium sulfate
Device Works, Shanghai, China) with a combined glass–calomel and removed under reduced pressure. Recrystallization from
1
electrode. Mass spectra were obtained using a Bruker maXis methanol yielded an orange solid (175 mg, 45%). The H NMR
ultrahigh resolution-TOF MS system. Fluorescence spectra were properties and findings are as follows: (400 MHz, DMSO-d6)
measured on an FLS-980 Edinburgh fluorescence spectrometer. d 11.21 (s, 1H), 8.46–8.14 (m, 3H), 8.06 (d, J = 8.7 Hz, 1H), 7.78
Cellular bioimaging was carried out on a confocal microscope (d, J = 8.2 Hz, 2H), 6.78–6.42 (m, 2H), 5.40 (s, 2H). 13C NMR
(Leica TCS SP8). Absorbance was measured with a microplate (101 MHz, DMSO-d6) d 166.6, 160.6, 153.9, 147.6, 144.2, 131.0,
reader (Synergy 2, Biotek, USA) in the MTT assay.
128.9, 124.1, 116.0, 114.9, 112.6, 110.3, 101.0, 74.8, 69.3, 40.6,
40.4, 40.2, 40.0, 39.7, 39.5, 39.3. HRMS calculated for
C17H11N3O4 [M À H]À m/z 320.0665, found 320.0701.
Synthesis of probe CNN
Synthesis of compound 1. 2,4-Dihydroxybenzaldehyde (2 g,
Synthesis of the CNN probe. Compound 3 (175 mg, 0.54 mmol),
14.5 mmol) and imidazole (1.15 g, 16.9 mmol) were dissolved in 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dienyl)butyric acid
dichloromethane, and stirred at room temperature for 5 min (150 mg, 0.6 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbonyl-
under an argon atmosphere, followed by addition of tert-butyl- diimide hydrochloride (157 mg, 0.8 mmol) were dissolved in
dimethylsilyl chloride (2.2 g, 14.5 mmol). The mixture was then dichloromethane and stirred at room temperature overnight. After
stirred at room temperature for 4 hours, and the progress of the the reaction was complete, the residue was washed with deionized
reaction was monitored by TLC. After the reaction was com- water, and the organic phases were combined. The organic phase
plete, the residue was washed with deionized water and brine, was dried with anhydrous sodium sulfate, and the solvent was
and the organic phases were combined. The organic phase was removed under reduced pressure. The residue was purified using
dried with anhydrous magnesium sulfate, and the solvent was silica gel column chromatography with PE/EA (10/1) to yield a light
1
removed under reduced pressure. The residue was purified yellow solid (178 mg, 59%). The H NMR properties and findings
using silica gel column chromatography with PE/EA (15/1), are as follows: (400 MHz, CDCl3) d 8.31 (d, J = 8.6 Hz, 2H), 8.25 (d, J =
yielding a colorless oily liquid (2.3 g, 63%). The 1H NMR 8.8 Hz, 1H), 8.19 (s, 1H), 7.59 (t, J = 10.7 Hz, 2H), 6.82 (dd, J = 8.7, 1.9
properties and findings are as follows: (400 MHz, CDCl3) Hz, 1H), 6.73 (d, J = 1.9 Hz, 1H), 5.22 (d, J = 11.9 Hz, 2H), 3.27 (s, 2H),
d 11.35 (s, 1H), 9.73 (s, 1H), 7.41 (d, J = 8.5 Hz, 1H), 6.54–6.34 2.19 (s, 3H), 1.94 (d, J = 9.6 Hz, 6H), 1.52 (s, 6H). 13C NMR (101 MHz,
(m, 2H), 0.99 (s, 9H), 0.28–0.25 (m, 6H). 13C NMR (101 MHz, CDCl3) d 190.6, 187.3, 170.3, 158.4, 156.6, 152.6, 150.9, 148.20, 142.5,
CDCl3) d 194.5, 164.1, 163.8, 135.4, 115.8, 113.0, 107.6, 77.4, 141.7, 139.9, 139.0, 130.2, 128.1, 124.2, 118.0, 115.5, 114.0, 112.7,
77.0, 76.7, 25.7–25.4 (3C), À4.1 to 4.4 (3C). HRMS, calculated for 106.4, 82.0, 77.3, 77.0, 76.7, 69.9, 58.4, 47.6, 38.4, 29.1, 18.4, 14.5,
C
13H20O3Si [M À H]À m/z 251.1097, found 251.1082.
12.6, 12.2. HR-MS, calculated for C31H27N3O7 [M + Na]+ m/z
Synthesis of compound 2. Compound 1 (2 g, 8 mmol) and 576.1741, found 576.1731.
silver oxide (7.4 g, 32 mmol) were added to anhydrous ether and
stirred for 5 min, followed by addition of 4-nitrobenzyl bromide
(8.6 g, 40 mmol). The reaction progress was monitored by TLC.
After the reaction was over, excess silver oxide was filtered off,
Conclusions
the solvent was removed under reduced pressure, and the In summary, a novel dual-site fluorescent probe, CNN, for the
residue was purified using a silica gel column with PE/EA(10/1), simultaneous detection of two potential cancer biomarkers,
This journal is ©The Royal Society of Chemistry 2019
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (21874085, 21535004, 91753111 and
21775091) and the Key Research and Development Program
of Shandong Province (2018YFJH0502).
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