Toward a chemical marker for inflammatory disease: A fluorescent probe for membrane-localized thioredoxin

Article abstract of DOI:10.1021/ja503356q

Thioredoxin (Trx) is a redox-active protein that plays a key role in mitigating the effects of oxidative stress. The secretion of Trx on the plasma membrane has been suggested as a distinctive feature of inflammation. However, selective monitoring of membrane-associated Trx activity has proved challenging because of the ubiquity of Trx action in cells. Here, we report a Trx-specific probe that allows visualization of Trx activity associated with the membranes via fluorescence microscopy. The ability of this probe to act as a possible screening tool for agents that modulate Trx secretion was demonstrated in HeLa cells under oxidative stress conditions and in a cellular hepatosteatosis model. Control experiments serve to confirm that the response seen for the present probe is due to Trx and that it is selective over various potentially competing metabolites, including thiol-containing small molecules and test proteins.

Full text of DOI:10.1021/ja503356q

Article
pubs.acs.org/JACS
Toward a Chemical Marker for Inflammatory Disease: A Fluorescent
Probe for Membrane-Localized Thioredoxin
Min Hee Lee,†,§ Hyun Mi Jeon,‡ Ji Hye Han,‡ Nayoung Park,§ Chulhun Kang,*,‡ Jonathan L. Sessler,*,†
and Jong Seung Kim*,§
†Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712-1224, United States
‡The School of East-West Medical Science, Kyung Hee University, Yongin 446-701, Korea
§Department of Chemistry, Korea University, Seoul 136-701, Korea
S
* Supporting Information
ABSTRACT: Thioredoxin (Trx) is a redox-active protein that plays a key role in
mitigating the effects of oxidative stress. The secretion of Trx on the plasma membrane has
been suggested as a distinctive feature of inflammation. However, selective monitoring of
membrane-associated Trx activity has proved challenging because of the ubiquity of Trx
action in cells. Here, we report a Trx-specific probe that allows visualization of Trx activity
associated with the membranes via fluorescence microscopy. The ability of this probe to act
as a possible screening tool for agents that modulate Trx secretion was demonstrated in
HeLa cells under oxidative stress conditions and in a cellular hepatosteatosis model.
Control experiments serve to confirm that the response seen for the present probe is due to
Trx and that it is selective over various potentially competing metabolites, including thiol-
containing small molecules and test proteins.
Membrane-localized Trx activity might thus be a good clinical
marker for the anti-inflammatory action of cells. So far, it has
been shown that most human cell lines have a membrane-
associated Trx, as inferred from indirect immunofluorescence
and Western blotting analyses.14−16 However, the activity of
membrane-localized Trx and the mechanism of its secretion in
association with an inflammatory insult are not fully under-
stood. Since the oxidation of Trx on the membrane triggers a
cellular inflammatory response, a readily available fluorescent
probe that would allow Trx activity to be monitored directly at
(or around) membrane sites is expected to be particularly
useful. The probe could cast new light on the mechanism of
Trx action during inflammation and allow the study of
inflammation-related disease via optical methods. Ultimately,
such a locus-specific Trx sensor system could allow for
diagnoses to be made without the need for invasive procedures.
Currently, we are unaware of any fluorescence probe that
may be used for the determination of membrane-associated Trx
activity. Trx probes are, however, known. For instance, we
previously reported a fluorescent probe that can visualize
mitochondrial Trx activity (as opposed to membrane-localized
Trx activity) in living cells.18 This system proved highly specific
for Trx. Thus, building off this prior work, we have developed a
new membrane-targeted Trx-specific fluorescent probe. As
detailed below, this new system, probe 1, acts as a chemical
marker for inflammation.
INTRODUCTION
■
Thioredoxin (Trx), along with Trx reductase and NADPH,
comprises an important component of the cellular antioxidant
system. Trx contains two adjacent Cys residues in its active site,
and their oxidation to the corresponding disulfide serves to
transfer reducing equivalents (re)supplied by Trx reductase and
NADPH. The Trx redox system plays a number of critical roles
in cells,1 including reduction of oxidized proteins,2 scavenging
reactive oxygen species (ROS),3 regulating cell signaling,4
controlling growth,5 and mediating both anti-apoptotic6 and
anti-inflammation7 functions. Its biological importance is
underscored by the fact that non-homeostatic Trx levels are
seen in several types of cancers,8 cardiovascular disease,9 and
diabetes,10 as well as in inflammation.11
Although Trx is relatively ubiquitous in cells, its expression at
specific locations may be implicated in different diseases. For
instance, increased Trx within the nucleus or in the cytosol has
been observed in the case of many cancers.12 In contrast, it has
been suggested that membrane-associated Trx may be an
indicator of inflammation.3,7,13−17 Having tests for Trx that are
specific to different subcellular locales could be useful in
discriminating between different Trx-releasing determinants
and thus differentiating between various disease states. This
could allow for improved diagnoses and obviate the need for
invasive procedures, such as tissue biopsies.
In the case of inflammation, Trx on the plasma membrane is
secreted to the extracellular medium in response to oxidative
stress, and the released Trx engenders cytoprotective effects
under oxidative stress and inflammatory conditions.3,7,13−17
Received: April 3, 2014
Published: May 19, 2014
© 2014 American Chemical Society
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the four hydrophilic carboxylic acid groups delay its subsequent
diffusion across the membrane. The disulfide-linked naph-
thalimide moiety18 is preferentially reduced by Trx with an
approximately 5000-fold faster reaction rate than that mediated
by GSH; this disulfide reduction and corresponding bond
cleavage provides an easy-to-monitor fluorescent signal at 540
nm, as illustrated in Scheme 1.
Scheme 1. Schematic Representation of the Reaction of 1
with the Membrane-Localized Trx
a
Probe 1 was prepared by the synthetic route outlined in
Scheme 2. Two references, compounds 2 and 12,18 without a
disulfide linkage and without the dodecyl alkyl chain and four
carboxylic acid groups, respectively, were also prepared for the
comparison (Scheme 2). Their chemical structures were
a
As detailed in the present report, probe 1 interacts with the lipid
bilayer of a cell membrane, where Trx-induced reduction of the
disulfide bond triggers a fluorescence change.
1
confirmed by H and 13C NMR spectroscopy, MALDI-TOF
mass spectrometry (MS), and ESI-MS (Figures S18−S47). The
synthesis of compounds 1−12 is described fully in the
Supporting Information.
The UV/vis absorption and fluorescence features of 1 were
studied in the presence of metabolic thiols, essential metal ions,
and hydrogen peroxide (H2O2) at 37 °C in phosphate-buffered
saline (PBS) at pH 7.4. Addition of 1 to PBS solutions
containing biological thiols, such as Trx, glutathione (GSH),
homocysteine (Hcy), and cysteine (Cys), led to a shift in the
absorption and emission bands of the probe from 376 and 495
RESULTS AND DISCUSSION
■
We report a fluorescent sensor (probe 1) that allows for the
selective visualization of membrane-associated Trx activity in a
cell-based inflammation model. As can be seen from an
inspection of its chemical structure (Schemes 1 and 2), probe 1
is composed of a disulfide-linked naphthalimide, a dodecyl alkyl
chain, and four carboxylic acid groups. The lipophilic alkyl
chain serves to guide the probe 1 to the cell membrane, while
a
Scheme 2. (a−c) Synthetic Routes to Compounds 5, 10, 1, and 2 and (d) Structure of the Reference Compound 12
a
TEA, triethylamine; DCM, dichloromethane; DMF, N,N-dimethylformamide; DIPEA, N,N-diisopropylethylamine; TFA, trifluoroacetic acid.
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to 430 and 540 nm, respectively (Figure S1). In contrast, no
such significant changes were observed upon exposure to other
putative analytes. Of note is that, in the presence of 5.0 μM Trx,
the fluorescence intensity (FI) of 1 at 540 nm was enhanced by
ca. 9-fold. This is a greater enhancement than seen in the
presence of 5 mM of other test biological thiols, viz. GSH, Cys,
and Hcy. Typical intracellular Trx levels (ca. 5−10 μM) are
roughly 1000-fold lower than those of other biological
thiols.18,19 Thus, the selectivity displayed by 1 was thought to
augur well for its use as a membrane-localized, Trx-specific
probe. Evidence in support of this postulate is provided below.
As a first step toward confirming the sensitivity, the
fluorescence spectra of 1 were recorded at various Trx
concentrations (0−5.0 μM). The induced changes were
found to reach a plateau upon the addition of 1.0 equiv of
the probe, and over this 0−1 equiv regime, a linear relationship
(R2 = 0.9853) between the FI at 540 nm and the Trx
concentration was observed (see Figure 1a,b). Based on a
glycero-3-phosphocholine (DPPC) and 40 mol% cholesterol
(cf. Figure 1c,d). The liposomes were prepared in accord with
literature procedures20 and characterized by transmission
electron microscopy (TEM) (Figure S4). In accord with
design expectations, Trx in solution was found to react with 1
embedded in the liposome and produce a large fluorescence
enhancement at 531 nm (Figure 1c); presumably, this
enhancement reflects disulfide bond cleavage in accord with
the scenario presented in Scheme 1. The maximum emission
wavelength in liposomes is slightly blue-shifted compared with
that in PBS solution. A linear relationship (R2 = 0.9826)
between the FI at 531 nm and the Trx concentration (0−1.0
equiv) was found (Figure S5). The fluorescence changes of 1
(1.0 μM) in the liposomal formulation were also monitored as
a function of time in the presence of 5.0 μM Trx. It was found
that the FI at 531 nm increases gradually and reaches a plateau
within 30 min (Figure 1d).
To provide support for the notion that 1 can be used to
detect cellular Trx pools with negligible interference from other
biologically relevant analytes in membrane-like environments,
the fluorescence change seen for 1 in the presence of other
potential interferants, including GSH, Cys, Hcy, H2O2, and
several test biologically relevant metal ions, was recorded in
liposomes. We found the spectral changes are similar to the
results shown in Figure 1, except that extent of cleavage (of
probe 1) and associated optical changes produced by GSH
decrease is reduced in liposomes. Trx-based activity remains
high (cf. Figure S6).
Using confocal microscopy, the fluorescence changes of
HeLa cells treated with 1 were monitored as a function of time
(1−60 min) (Figure S7). This was done in order to obtain
insights into the cellular loci where probe 1 undergoes thiol-
mediated disulfide cleavage. Results from these experiments are
presented in Figure 2a, which shows confocal microscopy
images obtained at 1, 5, and 20 min after incubation with the
probe. These images are displayed as green, red, and blue-
colored images, respectively, and are overlaid to give various
pseudo color images (1 + 5 min, 5 + 20 min, and 1 + 5 + 20
min, respectively). An intense fluorescence signature resulting
from the disulfide cleavage of 1 begins to appear around the cell
membrane by 1 min (see green color in the overlay image of 1
+ 5 + 20 min). This intensity subsequently diffuses into the
cytosol where it is apparent by minute 20.
In contrast to what is true for probe 1, compound 2, the
fluorescent product of the cleavage reaction of 1, shows a fast
cellular uptake into the cytosol of the cells without the
membrane associated fluorescence features seen in the case of 1
(Figure S8). On the basis of these observations, we suggest that
in the case of 1 disulfide bond cleavage occurs near the cell
membrane to generate a daughter product (fluorescent species
2), which then diffuses into the cytosol. In the case of 12, a
reference compound lacking a dodecyl alkyl chain and the four
carboxylic acid groups found in 1, similar experiments reveal a
near complete absence of membrane-associated FI (Figure S9).
To provide further support for the suggestion that a
membrane-associated fluorogenic disulfide bond cleavage
reaction involving 1 is followed by the endocytotic transfer of
the resulting fluorescent product (2) into the interior of the
cells, experiments analogous to the above were performed in
the presence of methyl-β-cyclodextrin (MCD). MCD is a
known inhibitor of caveolae-dependent endocytosis and its
effect is concentration dependent.21 It can thus be used to test
whether lipophilic compounds are being internalized into cells
Figure 1. Trx-induced changes in the photophysical properties of
probe 1 in PBS solution and liposomes. (a) Fluorescence changes of 1
(1.0 μM) observed upon treatment with increasing concentrations of
Trx (0−5.0 μM). (b) Change in the fluorescence intensity (FI) at 540
nm as a function of Trx concentration. Spectra in panels (a) and (b)
were acquired 30 min after addition of Trx and were recorded in PBS
solution (pH 7.4) containing 5% (v/v) of DMSO at 37 °C. (c)
Fluorescence spectra of 1 (5.0 μM) recorded in the absence (red line)
and presence (blue line) of Trx (5.0 μM). Black line represents
liposome (DPPC/40 mol% cholesterol) only. Data were acquired 30
min after mixing 1 with Trx in the liposome at 37 °C. (d) Time course
of the fluorescence response of 1 (1.0 μM) with Trx (5.0 μM) in
liposomes. Excitation was effected at 430 nm.
MALDI-TOF mass spectrometric analysis of the reaction
product, we suggest that under these conditions probe 1 is
converted to [2+H]+ (Figure S2). In addition, the absorption
and fluorescence spectra of the product produced when 1 is
exposed to Trx are fully consistent with those of 2 as prepared
through independent synthesis (Figure S3). On this basis, we
suggest that probe 1 has the sensitivity needed to be used as a
membrane-localized Trx sensor.
To test the above assumption in a hydrophobic environment
designed to model that of the cell membrane, probe 1 was
embedded into a liposome composed of 1,2-dipalmitoyl-sn-
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To confirm the site of initial fluorogenic reaction of probe 1,
colocalization experiments were carried out using a fluorescent
membrane tracker (DiIC12) and with an early endosome
tracker (Early Endosomes-RFP). As shown in Figure S10, the
fluorescence image of 1 partially overlaps with those produced
by the membrane tracker, and mostly at the cytosolic side of
the membrane (panels a−d in Figure S10). On the other hand,
a poor fluorescence overlap was seen in the colocalization
studies involving the early endosome tracker (panels e−h in
Figure S10). From these experiments, we infer that the thiol-
induced fluorogenic reaction of 1 (1) is membrane associated,
(2) involves predominantly thiols that are present on the
cytosolic side of the membrane and (3) is not dependent on an
endocytotic process.
To identify the thiol species responsible for the fluorogenic
reaction of 1 shown in Figure 2, the cells were treated with PX-
12, a selective Trx inhibitor.18,23 As can be seen from an
inspection of the upper panels of Figure 3, the FI of the HeLa
Figure 2. Confocal microscopy images of HeLa cells showing the
fluorogenic response as a function of time after incubation with probe
1 and the effect of methyl-β-cyclodextrin (MCD) on the cell
environment-induced changes in the fluorescence features of probes
1 and 12. (a) HeLa cells were incubated with PBS containing 1 (5.0
μM) at 37 °C. The images were obtained at time points consisting of
1, 5, and 20 min after the addition of 1. The intensities of the images
were then adjusted to be similar to allow for direct comparisons.
Finally, the images were merged, 1 + 5 min, 5 + 20 min, and 1 + 5 + 20
min. (b) Confocal microscopy images and overlay with the
corresponding differential interference contrast images of HeLa cells
treated with 1 and 12, respectively. The studies were carried out in the
absence and presence of MCD. Here, the cells were separately
pretreated with media containing MCD (0, 3, 5, and 10 mM) and
incubated for 1 h at 37 °C. The media were replaced with PBS
containing 1 (5.0 μM) or 12 (1.0 μM). After 5 min, the fluorescence
images were recorded.
Figure 3. Changes in the fluorescence emission features of 1 and a
control system (12) seen in HeLa cells and in protein extract. (a)
Effect of PX-12, a selective inhibitor of Trx, on the fluorogenic
response of 1 and 12 in HeLa cells. The cells were separately
pretreated with media containing PX-12 (0, 2, and 10 μM) for 24 h at
37 °C. The media were replaced with PBS containing 1 (5.0 μM) and
12 (1.0 μM), respectively. After incubation for 10 min at 37 °C, the
fluorescence images were recorded. The histogram is based on the
averaged fluorescence intensity (FI) of each cell in the displayed
images and shows the standard deviation as error bars (n = 5). (b)
Effect of removing Trx by immunoprecipitation (IP) on the
fluorescence of 1 (10.0 μM): before (crude) and after (IP
supernatant). Time course of the fluorescence change of 1 in the
protein extract (1.0 mg/mL). (c) Fluorescence enhancement of 1 in
crude and IP-S. The excitation and emission wavelengths were 485 and
535 nm, respectively. The immunoreactivity of crude and IP-S are
shown in a Western blot.
via the caveolae pathway.22 In accord with our hypothesis,
probe 1 was found to undergo a fluorogenic reaction regardless
of the presence or absence of MCD. On the other hand, the
fluorescent signal ascribed to daughter product 2 is observed
near the membrane in a MCD dose-dependent manner (cf.
upper panels in Figure 2b). Finally, the fluorogenic response
produced by control 12 was seen to decrease gradually as the
concentration of MCD increased (lower panels in Figure 2b);
this is expected for a system where disulfide cleavage takes place
predominantly within the cytosol. The contrasting behavior
between 1 and 12 is fully consistent with the proposition that
probe 1 targets the membrane-associated thiols to give a
fluorescence change as depicted in Scheme 1.
cells treated with 1 decreases in a PX-12 dose-dependent
manner. In contrast, the fluorescence from 12 is unchanged
under similar experimental conditions (lower panels in Figure
3). The removal of Trx from the crude extract of HeLa cells by
immunoprecipitation (IP) also significantly reduced the FI
ascribed to probe 1 and its environmental response (Figure
3b,c).
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As further controls, studies were carried out in the presence
of bacitracin, an inhibitor of protein disulfide isomerase
(PDI),24 and in the presence of E64, an inhibitor of other
Cys-containing proteins.25 In the case of both test experiments,
little in the way of fluorescent changes were observed for probe
1 (Figure S11). Together with the data presented in Figure 3,
we thus conclude that the disulfide bond cleavage reaction of 1
is predominantly caused by Trx in cells, and not by PDI, Cys-
containing proteins, or other cytosolic protein thiols. To the
extent this conclusion is correct, it leads to the suggestion that
probe 1 can be used to test for the presence of absence of Trx
at or near cell membranes, as well as its secretion from those
loci into the extracellular space in response to an inflammatory
insult.13 This hypothesis was further tested via a series of
experiments as detailed below.
A first set of studies involved testing a possible mode of Trx
action. Recently, it was suggested that the anti-inflammatory
effect of Trx involves a complex between Trx and TXNIP
(thioredoxin-interacting protein).26,27 According to this sugges-
tion, under conditions of oxidative stress, Trx is oxidized and
the TXNIP is released to bind to NLRP3 in an inflammasome;
this activates its inflammatory response, including IL-1β
secretion.28 To investigate whether the proposed Trx activity
shown in Figures 2 and 3 involves the Trx-TXNIP complex, the
fluorogenic reaction of 1 was monitored in the presence of
okadaic acid (OKA). OKA is an inhibitor of phosphoprotein
phosphatases.29 It reduces the concentration of TXNIP, which
in turn increases the activity of Trx.30,31 In the present study,
HepG2 cells were separately pretreated with OKA (0, 50, and
100 nM, respectively). The media were then replaced with fresh
media containing 5.0 μM of probe 1, and the images were
monitored as a function of time (recorded at 1, 5, 20, and 60
min after addition). As can be seen from an inspection of
Figure 4, the fluorogenic response of 1 gradually increases as
image). The image produced by Trx mainly overlaps with that
of TXNIP (panels a−e), but not with that of NLRP3 (panels f−
j). Also, as can be seen from an inspection of panels e and j in
Figure S12, the Z-stack orthogonal images are consistent with
the suggestion that the Trx overlaps with TXNIP in a focal
plane. In contrast, no overlap is seen with NLRP3. The results
provide further support for the previously stated conclusion,
namely that Trx forms a complex with TXNIP and, as a result,
these two species colocalize within the cell.
Having established that the fluorogenic response of 1 is
associated with Trx activity, we sought to determine whether it
could be used to signal the loss of membrane-associated Trx
upon secretion into the extracellular media in analogy to IL-1β
secretion under conditions of inflammatory stimulation. The
secretion of Trx can be increased by adding cycloheximide and
dinitrophenol.13 Therefore, the fluorescence response of 1 in
HeLa cells, which has Trx on the cell membrane, was
monitored in the presence of cycloheximide and dinitrophenol.
The results are summarized in Figure 5. As can be seen from an
Figure 5. (a) Fluorescence change of 1 (5.0 μM) observed in HeLa
cells treated with the Trx secretion activators cycloheximide (0.1 mM)
and dinitrophenol (0.5 mM) for 3 h at 37 °C. Bars represent the
averaged fluorescence intensity (FI) of each cell in confocal images
recorded 1, 2, 3, 5, 10, 20, 30, and 60 min post treatment. (b) Western
blot shows bands corresponding to the Trx (12 kDa) of cell culture
medium in the absence and presence of cycloheximide or
dinitrophenol. The histograms are based on the average and show
the standard deviation as error bars (n = 4). Statistical significance is
marked with a * when p < 0.05.
Figure 4. Fluorescence change of 1 in HepG2 cells treated with
okadaic acid (OKA). Cells were separately pretreated with media
containing OKA (0, 50, and 100 nM, respectively) for 1 h at 37 °C.
The media were replaced with PBS containing 1 (5.0 μM). The images
were then obtained at time points consisting of 1, 5, 20, and 60 min
post addition at 37 °C. The histogram shows the averaged
fluorescence intensity (FI) of each cell in the displayed images.
inspection of this figure, a reduced fluorescence response
relative to untreated cells is seen after treatment with
cycloheximide or dinitrophenol over the 1−60 min time
range. This result is consistent with loss of membrane-localized
Trx due to secretion over this time period (cf. Figures 5a and
S13). As the incubation time increases, however, restoration of
fluorescence is observed; presumably, this reflects regeneration
of membrane-localized Trx and its reaction with unreacted
probe 1.
the concentration of OKA increases. This leads us to suggest
that the increased fluorogenic response produced by 1 reflects
the increase in Trx activity produced upon OKA inhibition of
TXNIP.
To confirm the presence of a Trx-TXNIP complex in HepG2
cells, colocalization experiments with Trx, TXNIP, and NLRP3
were carried out using immunofluorescence analysis. Figure S12
displays the confocal immunofluorescence images, their overlay
images, and the corresponding Z-stack orthogonal images (3D
Confirmation that Trx is in fact secreted into the extracellular
medium came from Western blot analyses of the cell culture
media (Figure 5b). As expected, enhanced secretion of Trx was
observed from the cells treated with cycloheximide or
dinitrophenol. This provides further support for the conclusion
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that probe 1 and its fluorogenic response may be used to
monitor the induced secretion of Trx in vitro. Consistent with
this supposition is the finding that compound 12 undergoes
little change in its fluorescence character in cellular media
containing cycloheximide or dinitrophenol (Figure S14).
Similar experiments were also performed with the mitochon-
drial Trx-responding probe that we previously reported.18 Here
again, and in marked contrast to what was seen for 1, little
fluorescence change was observed under conditions favoring
the secretion of Trx (Figure S15).
To test whether probe 1 might have utility as a potential
diagnostic of inflammation, a hepatotoxic cellular model for
fatty liver disease was used.32 In these tests, HepG2 cells were
treated with palmitic acid (PA) and oleic acid (OA). An excess
of PA is known to induce oxidative stress,33 endoplasmic
reticulum stress,34 mitochondrial malfunction,35 and inflamma-
tion36 in HepG2 cells. The underlying cytotoxic process is
considered to be similar to hepatosteatosis, a condition known
as fatty liver disease.37 In contrast, OA is less cytotoxic toward
HepG2 cells.38 As seen in confocal images and the
corresponding histograms of Figure 6a, PA-treated cells
containing 1 produce a diminished fluorescence response
near the cell membrane, while OA-treated cells are charac-
terized by a fluorescence that matches that of cells containing
only the probe. Moreover, an increase in the anti-Trx
immunoreactivity was observed in the case of PA treatment
relative to what was seen for the untreated group or cells
exposed to OA (Figure 6b). Confocal imaging (cf. Figure 6c)
provided further support for the notion that the anti-Trx
immunoreactivity was decreased in the case of the PA-treated
cells as compared to the untreated or the OA-treated cells (see
also Figure S16).
Finally, the fluorogenic activity of 1 was checked in the HT-
29 cell line (Figure S17). Again, a Trx-induced fluorescence
increase was found around the cell membrane. Based on these
findings, we conclude that 1 could have a role to play as a probe
that is used to screen drug candidates, including potential anti-
inflammatory agents, which mediate the effects of Trx secretion.
The contribution to the fluorogenic reaction of probe 1 by the
secreted Trx in plasma and serum would be anticipated to be
negligible since in these loci [Trx] is in the nanomolar range39
and in a relatively more-oxidized redox state than intracellular
Trx.40 Thus, we suggest that probe 1 and other rationally
designed, site-selective Trx probes could be useful to play in
differentiating between various diseases associated with
oxidative stress without the need for invasive procedures such
as tissue biopsies.
Figure 6. (a) Fluorescence change of 1 observed in HepG2 cells
treated with fatty acids as a model of hepatosteatosis. Cells were
separately pretreated with 0.7 mM of palmitic acid (PA) and oleic acid
(OA) for 24 h at 37 °C. Fluorescence images of 1 were recorded at the
following time points: 1, 5, 20, 60 min. The histogram shows the
averaged fluorescence intensity (FI) of each cell in the images. (b)
Western blot shows bands corresponding to the Trx (12 kDa) of cell
culture medium in the absence and presence of PA or OA. The
histograms are based on the average and show the standard deviation
as error bars (n = 4). Statistical significance was marked with a * when
p < 0.05. (c) Confocal immunofluorescence images of the fixed
HepG2 cells (untreated, PA-treated, OA-treated cells) with antibodies
for Trx (green) and NLRP3 (red). Fluorescence Z-stack orthogonal
images were collected at 1 μm intervals ranging from 0 to 9 μm along a
Z-optical axis.
CONCLUSIONS
■
Reported here is a potential chemical marker for inflammatory
disease, probe 1. This membrane-targeting system produces a
highly selective fluorescent response in the presence of Trx but
not other biological thiols or other potential interferants.
Confocal microscopic studies with the HepG2 cell line provide
support for the design expectation that 1 interacts preferentially
with the lipophilic cell membrane and produces a strong
fluorescence response in the presence of membrane-associated
Trx. This fluorogenic response may be regulated via formation
of a Trx-TXNIP complex at or near the cell membrane. The
effects of dinitrophenol and cycloheximide, which serve to
enhance Trx secretion into the extracellular medium, could be
readily monitored using 1 as a probe in studies involving HeLa
cells. The potential utility of 1 as an indicator of pathogenic
states and as a possible screening tool for agents that can
manipulate secretion of membrane-associated Trx was demon-
strated using a fatty liver disease model in HepG2 cells.
Preliminary studies confirmed that 1 is also active as a Trx
probe in the HT-29 cell line. On the basis of these findings, we
suggest that this system could have a role to play in the
screening of new potential anti-inflammatory drugs and in the
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System, iNtRON, Inc., Kyunggi-do, Korea), followed by visualization
on X-ray film (AGFA). The density ratio was normalized to that of the
control. The values are expressed as mean SD, with an n = 4 (i.e.,
four independent samples from different cells). Data were analyzed
statistically by the Student’s t test, and a value of p < 0.05 was
considered to be statistically significant.
diagnosis and staging of inflammation-related disease via optical
methods that obviate the need for invasive procedures.
EXPERIMENTAL SECTION
■
Synthetic and Spectroscopic Methods. A complete listing of
the methods used to prepare and characterize all new compounds,
including probe 1, is included in the Supporting Information.
Liposome Preparation. Liposomes were prepared by the solvent
evaporation method.20 Briefly, 36 μL of (1,2-dipalmitoyl-sn-glycero-3-
phosphocholine) DPPC (0.1 M in chloroform) and 24 μL of
cholesterol (0.1 M in chloroform) were added to a 50 mL round-
bottom flask containing 940 μL of chloroform and 200 μL of
methanol. The aqueous phase (7 mL of HEPES buffer, 10 mM, pH
7.4) was then carefully added along the flask walls. The organic
solvents were removed in a rotary evaporator under reduced pressure
at 40 °C and 40 rpm. Subsequently, the resulting aqueous solution was
subjected to sonication for 30 min. The liposomes were characterized
by TEM (Figure S4). To stain the liposomes, a small amount of 1 in
DMSO was added with measurements being made after 1 h at 37 °C.
The [1]:[liposome] ratio was around 1:170.
Immunohistochemistry-Based Colocalization of Trx with
TXNIP or NLRP3. Cells in PBS solution were fixed with 4%
paraformaldehyde in PBS for 30 min at room temperature and washed
with a buffer (0.1% BSA and 0.1% Triton X-100). The fixed cells were
blocked with a blocking buffer (1% BSA). After the blocking buffer was
discarded, the cells were incubated with rabbit anti-Trx antibody
(1:200, diluted with 0.1% BSA) overnight at 4 °C. After removal of the
unbound antibody, the cells were incubated with goat anti-TXNIP or
mouse anti-NLRP3 antibodies (1:200, diluted with 0.1% BSA) for 1 h
at room temperature, followed by washing with the above buffer 3
times. The cells were incubated with the corresponding fluorescent
secondary antibodies (1:1000, diluted with 0.1% BSA) for 1 h at room
temperature. Finally, after washing, the fluorescence images of the cells
were obtained based on a Z-stack (3D image stack) using a confocal
microscope (Zeiss LSM 510 model) where the Z-stack images were
collected at 1 μm intervals over a 0−9 μm range. Merged Z-stack and
orthogonal images were then obtained. Trx (ab26320, Abcam, Inc.,
MA), TXNIP (SC-33099, Santa Cruz, Inc., TX), and NLRP3 (Cryo-2,
Adipogen, Inc., Incheon, Korea) were detected using 488 donkey anti-
rabbit IgG, 546 rabbit anti-goat IgG, and 633 goat anti-mouse IgG (A-
21206, A-21085, and A-21050 from Molecular Probes, Inc., OR)
antibodies. The images corresponding to Trx (green), TXNIP (red),
and NLRP3 (red) were obtained using excitation wavelengths of 488,
543, and 633 nm and 505−530 nm band-path, 560 nm long-path, and
650 nm long-path filters, respectively.
Cell Culture and Imaging. A human cervical cancer cell line
(HeLa) and a human hepatoma cell line (HepG2) were cultured in
Dulbecco’s Modified Eagle’s Medium (DMEM) and in RPMI 1640,
respectively, each supplemented with 10% FBS (WelGene), penicillin
(100 units/mL), and streptomycin (100 μg/mL). Two days before
imaging, the cells were placed on glass-bottomed dishes (MatTek)
which were incubated in a humidified atmosphere containing 5% (v/v)
CO2 at 37 °C. Cell images were obtained using confocal microscopes
from Leica (Leica model TCS SP2) and Zeiss (Zeiss model LSM 510).
All fluorescence images of probe 1 were obtained using an excitation
wavelength of 458 nm and a long path (>505 nm) emission filter.
Other information is available in the Figure captions.
Treatment of HeLa Cells with Cycloheximide and Dinitro-
phenol. For the tests of Trx secretion from HeLa cells, the cells were
plated at the 2 × 105/well level in glass-bottom dishes. After
incubation overnight, the cells were treated with DMEM containing
0.5 mM dinitrophenol or 0.1 mM cycloheximide for 3 h at 37 °C. The
media were exchanged for PBS containing 1 (5.0 μM) prior to
confocal microscopic imaging.
Treatment of HpG2 Cells with Fatty Acids. HepG2 cells were
plated at 2 × 105/well in glass-bottom dishes and incubated for 24 h.
The media were changed to one containing 0.7 mM OA or 0.7 mM
PA with bovine serum albumin (BSA) for 24 h at 37 °C. The media
were then replaced with RPMI (Roswell Park Memorial Institute)
medium without FBS for 4 h. Finally, the cells were incubated with
PBS containing 1 (5.0 μM) prior to confocal microscopic imaging.
Western Blot Experiments. To collect Trx protein that was
presumably secreted into the extracellular medium, media from each
well were treated with 20% trichloroacetic acid for 30 min in ice and
centrifuged at 14 000 rpm for 15 min at 4 °C. The resulting pellet was
washed with 200 μL cold acetone and centrifuged two more times
before being dried for 10 min by exposure to the laboratory
atmosphere to remove the acetone. After being air-dried in this way,
the pellet was resuspended in a sodium dodecyl sulfate−
polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The
secreted Trx was separated by SDS-PAGE (15% polyacrylamide) and
blotted onto a PVDR membrane (pore size 0.2 μm) using a Bio-Rad
Transblot kit (Trans-Blot SD semi-dry transfer cell kit, Bio-Rad, Inc.,
CA). The membrane was treated with a blocking solution (5% (w/v)
nonfat dry milk in TBS-T) for 1 h at room temperature, before being
subjected to primary antibody binding using goat anti-Trx antibody
(ab16965, Abcam, Inc., MA); this was done using a blocking solution
and overnight treatment at 4 °C. The excess antibody was removed by
rinsing three times with TBS-T. For the secondary antibody binding
studies, the membrane was incubated with a donkey anti-goat IgG-
HRP antibody (sc-2005, Santa Cruz, Inc., Texas) in a blocking
solution for 1 h at room temperature. After the membrane was rinsed
with TBS-T buffer 3 times, the immunoreactive bands were detected
via treatment with an ECL substrate solution (Western Blot Detection
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic and spectroscopic methods, additional spectra (UV/
vis absorption, fluorescence, NMR, ESI-MS, and MALDI-TOF
MS), and imaging data. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
kangch@khu.ac.kr
■
sessler@cm.utexas.edu
jongskim@korea.ac.kr
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by National Research Foundation of
The Ministry of Science, ICT & Future Planning in Korea (No.
2009-0081566, J.S.K.; NRF-2012R1A6A3A03037981, M.H.L.),
Cooperative Research Program for Agriculture Science &
Technology Development (No. PJ008959, C.K.), the U.S.
National Institutes of Health (CA 68687, J.L.S.), and the
Robert A. Welch Foundation (F-1018, J.L.S.).
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