Fluorescence imaging of selenol in HepG2 cell apoptosis induced by Na2SeO3

Article abstract of DOI:10.1039/c4cc06359g

A novel fluorescence probe (HB) has been designed and synthesized to image selenol in living cells and in vivo for the first time, and used to investigate the Na₂SeO₃ anticancer mechanism in HepG2 cells. This journal is

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ARTICLE TYPE
Fluorescence Imaging for Selenol in HepG2 Cell Apoptosis Induced by
Na2SeO3
Fanpeng Konga, Bo Hua, Yan Gaoa , Kehua Xu
*
a, Xiaohong Pana, Fang Huanga, Qiuling Zhengb, Hao Chenb and Bo
a
Tang
*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
In our previous work, we have found that the SeꢀN bond inꢀ
A novel fluorescence probe (HB) has been designed and
synthesized to image selenol in living cells and in vivo for the
first time, and been used to investigate the Na2SeO3
volved in ebselen can be cleaved by sulfhydryl group with high
50 selectivity, and developed some fluorescence methods to monitor
intracellular thiols and the changes of redox state.14ꢀ16 2,1,3ꢀ
benzoselenadiazole (BS) is also a containing SeꢀN compound, but
the SeꢀN bond should be stronger than that of ebselen, which has
been confirmed by calculating the Wiberg bond indices (WBI) of
55 BS and ebselen in Figure S1. The result shows that the WBI of
SeꢀN in BS is larger than that of ebselen (1.145 vs 0.832),
indicating that BS has less reaction activity than ebselen in the
presence of nucleophilic reagent. Thus we can assume that it
needs a stronger nucleophilic reagent to cleave the SeꢀN bond in
60 BS. Maybe, BS could discriminate between selenol and thiol,
because selenol group (ꢀSeH) has higher nucleophilicity than
thiol group (ꢀSH) does.17,18 Soon afterwards, we tried the idea and
found that BS can indeed react rapidly with Sec, but not with
Cys, in 0.01 M, pH 7.4 phosphate buffer solution (PBS), as
65 shown in Figure S3ꢀS4. Based on this, we designed a novel
fluorescent probe, 2ꢀ(benzoselenadiazolꢀ5ꢀyl)vinyl)styryl)ꢀ1ꢀ
ethylꢀ3,3ꢀdimethylꢀindolium iodide (HB), for probing
intracellular Sec in living cells and in vivo (Scheme 1). The probe
shows highly selective and sensitive to Sec in physiological
70 conditions and is able to monitor the changes of selenol level in
living cells and in vivo. Furthermore, we have observed that
selenol concentrations increase gradually in HepG2 cells induced
by Na2SeO3 in a timeꢀ and doseꢀdependent manner, with
accompanying tumor cell apoptosis, cell cycle arrest and
75 depletion of mitochondrial membrane potential.
10 anticancer mechanism in HepG2 cells.
Selenium (Se) is an essential trace element involved in different
physiological functions of the human body.1 More importantly,
the protective ability of Se against cancer was suggested in 1969
with the discovery of an inverse relationship between cancer
15 occurrence and Se content of local crop in the USA.2 There are
several proposed mechanisms to explain the effect of Se in cancer
prevention and treatment, such as apoptosis, autophagy, necrosis,
mitotic catastrophe or combinations thereof.3,4 But the exactly
mechanism of Se compounds for anticancer property remains still
20 unclear. It is suggested that the efficacy of Se in cancer
prevention and treatment is dependent on the chemical speciation
of Se.2,5 Thus there is a clear need to identify different Se species
in vivo in order to investigate their biological functions. The low
molecular weight selenium compounds, such as methylselenol6a,6b
25 and selenocysteine (Sec) are the key metabolites and found to be
important in cancer prevention.6cꢀ6d Especially, Sec as an
important low molecular weight selenniumꢀcontaining amino
acid, is located in the active sites of selenoproteins (SePs) and
encoded by a UGA stop codon.7 Sec is also the metabolic product
30 of Na2SeO3 and plays an important role in cancer treatment. In
order to investigate the physiological function of Sec and clarify
the anticancer mechanism of selenium, there is a pressing need to
develop a method to monitor Sec in living cells and in vivo.
Fluorescence probes with high sensitivity, good selectivity, short
35 response time, and advantage of direct observation, are powerful
tool for the identification of substances in biologial systems.8ꢀ11
Up to now, very few fluorescence probes for detecting the Sec or
selenoprotein12 have been reported. In 2006, Maeda et al.
reported a fluorescent probe BESThio to discriminate between
40 Sec and Cys, based on the difference that the selenol group of Sec
exists in the moreꢀreactive, ionized form, whereas the thiol group
of Cys is protonated and less reactive at pH 5.8.13 Unfortunately,
it was not suitable for detecting or imaging Sec at physiologic
conditions. So, it is still a challenge to develop a fluorescence
45 method to detect Sec in living cells and tissues to gain insight into
the physiological functions of Sec and the inhibitory mechanism
of Se to tumor cells.
Scheme 1. (a) A novel reporter group for Sec; (b) Design strategy of
fluorescent probe (HB)
80
The fluorescence probe (HB) was synthesized by integrating the
BS moiety into a hemicyanine dye via Knoevenagel condensation
and Wittig reaction in moderate yields (Scheme S1), and the
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structure of HB was confirmed by 1H NMR, 13C NMR and
HRMS (see ESI). Here, a hemicyanine dye was chosen, not only
because its positive charge can improve the reaction of HB with
Sec and increase the water solubility of HB, but also because an
obvious change in fluorescence intensity before and after addition
of Sec into the probe solution may be observed. That is, when the
BS moiety in HB is transformed into diamine product, the double
electronꢀacceptor structure of HB will become an electronꢀdonor
and electronꢀacceptor form of product, which would be very
from 10 ꢁM of (CysSe)2 and 20 ꢁM of Cys in H2O for 30 min. (b) The
50 relative fluorescence responses of 10 ꢁM HB to thiols and other sulfur
compounds (10 mM for GSH, 2.5 mM for Cys and NAC, 1.0 mM for βꢀ
ME, 20 ꢁM for DTT, 50 ꢁM for H2S, 10 ꢁM for Na2SeO3, and 2.5 ꢁg/mL
for TrxR. Black bars represent the addition of one of these interferences to
a 10 ꢁM solution of HB. Red bars represent the addition of Sec plus one
55 of these interferences to the probe solution (*Sec was replaced by 60 ꢁM
of GSH). All data were acquired in 10 mM PBS buffer with pH 7.4 at
37°C (λex /λem = 460/ 580 nm).
5
To study the selectivity of HB toward Sec, we tested its responses
60 toward thiols, thioredoxin reductase (TrxR), NꢀacetylꢀLꢀcysteine
(NAC), hydrogen sulfide (H2S), Na2SeO3 and a mixture of
Na2SeO3 and GSH. The reducing reagent betaꢀmercaptoethanol
(βꢀME), dithiothreitol (DTT), Cys and glutathione (GSH) were
examined as the thiols. The addition of Cys is essential not only
65 because its content is rich in living cells but also because Sec is
generated from (CysSe)2 and Cys. As expected, HB exhibited an
obvious increase in fluorescence intensity upon reaction with Sec
or a mixture of Na2SeO3 and GSH (GSeH was generated19),
whereas the responses toward thiols and others, or even TrxR
70 were negligible (Figure 2b). One probable reason for no response
of HB to TrxR is that the Sec residues in the SePs are buried too
deeply to react with HB (Figure S8). In addition, the interference
from metal ions, reactive oxygen species (ROS), amino acids and
vitamin C were also studied, as shown in Figure S8. HB
75 displayed no fluorescent response toward the aboveꢀmentioned
bioanalysts (An error of < ± 5.0% in the relative fluorescence
intensity was considered tolerable).
We applied HB to visualize the intracellular Sec. Cells were
divided into four groups. One group was chosen as control, and
80 the other three groups were treated with Cys, selenocystine
(CysSe)2, and Sec generated by (CysSe)2 plus Cys, respectively.
Then the in situ changes of fluorescence were realꢀtimely
observed. Figure 3aꢀb showed that the Secꢀtreated cells emitted
immediately bright fluorescence, while the fluorescence signal
85 derived from the (CysSe)2ꢀtreated cells increased gradually until
1.5 h. The result reveals that the probe could monitor the dynamic
changes of Sec level, and (CysSe)2 could be transformed into Sec
in living cells. We next assessed the ability of HB to image Sec in
a mouse model20 (Figure S12), and the results indicated that the
90 probe was capable of detecting Sec in vivo.
10 beneficial to emit a bright fluorescent signal. The reaction
products of HB and Sec were fully characterized by mass
spectrometry and the collisionꢀinduced dissociation (CID) MS2
spectrum. (Figure S5).
The interaction between HB and Sec was first studied in
15 phosphate buffer (10 mM, pH 7.4, 1% DMSO) at room
temperature by emission spectroscopy. Excitation and emission
spectra were recorded during the reaction of HB with Sec (Figure
S6). HB shows an emission of 580 nm with a large Stokes shift of
120 nm. Optimal conditions of the response of HB to Sec were
20 worked out as shown in Figure S7, including pH and
concentration of HB. In order to apply in biological system,
physiological pH (7.4) was selected for the following
determination system. A gradual increase in the fluorescence
intensity was observed with adding different concentrations of
25 Sec (The details of preparing the stock solution of Sec see ESI
part 3) into 10 ꢁM of probe solution under optimal conditions
(Figure 1a), and the fluorescence quantum yield increased from
0.019 to 0.110 upon addition of Sec to HB. There was a good
linearity between the fluorescence intensities and Sec
30 concentrations in the range 0ꢀ20 ꢁM (Figure 1b). The regression
equation was F = 104.9 + 14.7 [Sec] ꢁM with a linear coefficient
of 0.9942. The limit of detection was 7.0 nM (standard deviation
3.5%, n = 11).
(a)
(b)
400
320
240
160
80
300
200
100
0
[Sec] ꢀΜ
500 550 600 650 700 750
Wavelength / nm
0
5
10
[Sec] / ꢀΜ
15
20
35 Fig.
1 (a) Fluorescence responses of 10 ꢁΜ HB to different
concentrations of Sec (0, 3, 6, 9, 11, 13, 15, 20 ꢁM).(b) A linear
correlation between emission intensities and concentrations of Sec. All
spectra were acquired in 10 mM PBS buffer with pH 7.4 at 37°C (λex /λem
= 460/ 580 nm).
40
Furthermore, a fast response of HB to Sec was confirmed by a
kinetics experiment, in which the fluorescence intensity instantly
increased by adding Sec into the probe solution and the intensity
was kept on a relative stable level for at least 30 min, as shown in
95 Fig. 3 (a) The in situ fluorescence changes of HB (10 ꢁM) response to Sec
with time in HepG2 cells. HepG2 cells were treated with 20 ꢁM of Cys,
20 ꢁM of (CysSe)2, and Sec derived from 10 ꢁM of (CysSe)2 and 20 ꢁM
of Cys for 10 min, respectively. The fluorescence changes of cells were
detected in 0 h and 1.5 h under confocal microscopy. (b) The fluorescence
100 intensity was quantified based on the results of the relative fluorescence
intensity of per cell in the scanned area.
45 Figure 2a.
(a) 400
(b)
200
100
0
*
300
200
Sodium selenite (Na2SeO3) is often used as anticancer reagents in
cancer treatment.21,22 However, how Na2SeO3 intersects with
100
0
5
10 15 20 25 30
Time / min
cancer development and therapeutic response is controversial3,4
.
Fig. 2 (a) The time courses of the fluorescence intensities of 10 ꢁM HB
with 20 ꢁM Sec (red line) and without Sec (black line). Sec was generated
105 Because Sec is the important metabolic product of Na2SeO3, it
2
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would be beneficial to reveal the exactly anticancer mechanism of
Na2SeO3 by investigating the change of selenol level in vivo and
the relationship between Sec and apoptosis or cell cycle. Firstly,
we have realꢀtimely observed the difference of selenol
min at 37°C in the dark for MMP. The results were displayed in
Figure 5b and Figure 5d. Mitochondrial membrane potential
decreases significantly with the increasing concentration of
sodium selenite (Figure 5b), and selenol level rises gradually and
5
concentrations between normal cells and cancer cells response to 50 reaches a maximum at 5 ꢁM of sodium selenite for 12 h (Figure
sodium selenite, as shown in Figure 4a. HepG2 and HLꢀ7702
cells were exposed to 5 ꢁM of sodium selenite for different time
(0ꢀ24 h) and then were cultured with the probe for 15 min. The
nuclei were stained by DAPI to show apoptosis process. A further
5d). Consequently, according to the above data, a potential
mechanism of Se anticancer could be suggested that sodium
selenite can be rapidly converted into high active selenol in
HepG2 cells, accompanying tumor cell cycle arrest and
10 experiment was carried out with different concentrations of 55 mitochondrial membrane potential collapse.
sodium selenite (0ꢀ10 ꢁM) for 12 h in Figure 4b. These results
show that the concentrations of selenol in HepG2 cells are higher
than those of HLꢀ7702 cells under the same experimental
conditions, and the apoptosis appears in HepG2 cells induced by
15 5 ꢁM of sodium selenite after 24 h or 10 ꢁM of sodium selenite
after 12 h (see the nucleiꢀstained blue images and AnnexinVꢀ
FITC/PI assay in Figure S13).
Fig.
5 Relationships between cell cycle distribution/ depletion of
60 mitochondrial membrane potential and Sec level. (a) HepG2 cell cycle
distribution, exposed to 5 ꢁM of selenite for 0ꢀ24 h, was detected by flow
cytometry. (b) HepG2 cells treated with different concentrations of
selenite for 12 h and then stained with the mitochondriaꢀselective JCꢀ1
dye were analyzed by flow cytometry. The values in the top right quadrant
65 of each dot plot represent the percentage of cells that emit green
fluorescence due to the JCꢀ1 monomers. (c) The corresponding changes
of fluorescence in (a) were detected by flow cytometer with CellQuest
analysis software. (d) The corresponding changes of fluorescence of (b)
were obtained by flow cytometer with CellQuest analysis software.
20
Fig. 4 (a) The in situ fluorescence changes of HepG2 and HLꢀ7702 cells
exposed to 5 ꢁM sodium selenite for different time (0ꢀ24 h) and then
incubated by HB (10 ꢁM). The nuclei were stained by DAPI. (b) The in
situ fluorescence changes of HepG2 and HLꢀ7702 cells exposed to
25 different concentrations of sodium selenite (0ꢀ10 ꢁM) for 12 h. The nuclei
were stained by DAPI.
70
Conclusions
The relation between cell cycle distribution and selenol level was
studied. HepG2 cells were exposed to 5 ꢁM of sodium selenite up
30 to different time (6ꢀ24 h), then the cells were harvested and
divided into two equal groups. One group was stained with 10
ꢁM of HB for 15 min and the fluorescence intensity of cells was
detected using flow cytometer. The other group was fixed with
70% ethanol overnight at 4°C for cell cycle distribution analysis.
35 The results show that the number of G1 phase cells increased
gradually and the S phase cells reduced correspondingly (Figure
5a), which is accompanied by a higher selenol concentration
(Figure 5c). Besides, we observed that the depletion of
mitochondrial membrane potential (MMP) induced by sodium
40 selenite had a close relationship with selenol level. HepG2 cells
were exposed to different concentrations of sodium selenite (2ꢀ10
ꢁM) up to 12 h. One group was stained with 10 ꢁM of HB for 15
min and the fluorescence intensity was detected using flow
cytometer. The other group was incubated with 10 ꢁM of JCꢀ1 (a
45 commercial probe for mitochondrial membrane potential) for 30
In summary, in order to investigate the Na2SeO3 anticancer
mechanism, we have designed and synthesized
a novel
fluorescence probe (HB) by integrating the BS moiety into a
75 hemicyanine dye, for the first time. The probe is highly sensitive
and selective for Sec over thiols and other bioactive species such
as essential ions and amino acids under physiological conditions.
HB has been successfully applied to image the concentration
changes of Sec in living cells and in vivo. Furthermore, combined
80 with the analysis of cell cycle and mitochondrial membrane
potential, we found that the HepG2 cells apoptosis induced by
Na2SeO3 was closely related with the selenol levels. The results
would be beneficial to improve the efficacy and expend the
application generality of Se compounds in cancer treatment and
85 prevention.
Acknowledgements
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This work was supported by 973 Program (2013CB933800); NSFC (Nos.
70
21227005, 21390411, 91313302, 21275092, 21328502, 21405098);
SDSFC (ZR2011BZ006), Specialized Research Fund for the Doctoral
Program of Higher Education of China (20123704110004) and Program
for Changjiang Scholars and Innovative Research Team in University.
HC thanks the support from NSF Career Award (CHE1149367).
5
75
Notes and references
a College of Chemistry, Chemical Engineering and Materials Science,
Collaborative Innovation Center of Functionalized Probes for Chemical
10 Imaging in Universities of Shandong, Key Laboratory of Molecular and
Nano Probes, Ministry of Education, Shandong Provincial Key
Laboratory of Clean Production of Fine Chemicals, Shandong Normal
University, Jinan 250014, P. R. China
b Department of Chemistry and Biochemistry, Ohio University, Athens,
15 Ohio 45701, USA
† Electronic Supplementary Information (ESI) available: [Details of
general experimental section; MTT assay; Cell cycle analysis;
Mitochondrial membrane potential analysis; 1H NMR, 13C NMR and
HRMS spectrum of intermediate products and HB]. See
20 DOI: 10.1039/b000000x/
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