An off-on COX-2-specific fluorescent probe: Targeting the golgi apparatus of cancer cells

Article abstract of DOI:10.1021/ja4056905

Identifying cancer cells and quantifying cancer-related events in particular organelles in a rapid and sensitive fashion are important for early diagnosis and for studies on pathology and therapeutics of cancers. Herein a smart off-on cyclooxygenase-2-specific fluorescence probe (ANQ-IMC-6), able to report the presence of cancer cells and to image Golgi-related events, has been designed and evaluated. Cyclooxygenase-2 (COX-2) has been used as imaging target in the probe design, since this enzyme is a biomarker of virtually all cancer cell lines. In the free state in aqueous solution, ANQ-IMC-6 mainly exists in a folded conformation where probe fluorescence is quenched through photoinduced electron transfer between the fluorophore acenaphtho[1,2-b]quinoxaline (ANQ) and the recognition group, indomethacin (IMC). Fluorescence is turned on, by restraining the photoinduced electron transfer, when ANQ-IMC-6 is forced to adopt the unfolded state following binding to COX-2 in the Golgi apparatus of cancer cells. ANQ-IMC-6 provides high signal-to-background staining and has been successfully used to rapidly differentiate cancer cells from normal cells when using flow cytometry and one- and two-photon fluorescence microscopic imaging. Furthermore, ANQ-IMC-6 may be able to visualize dynamic changes of the Golgi apparatus during cancer cell apoptosis, with possible application to early diagnosis.

Full text of DOI:10.1021/ja4056905

Article
pubs.acs.org/JACS
An Off−On COX-2-Specific Fluorescent Probe: Targeting the Golgi
Apparatus of Cancer Cells
Hua Zhang,†,§ Jiangli Fan,*,†,§ Jingyun Wang,‡ Shuangzhe Zhang,† Bairui Dou,‡ and Xiaojun Peng*,†,§
‡
†State Key Laboratory of Fine Chemicals and School of Life Science and Biotechnology, Dalian University of Technology, 2
Linggong Road, Hi-tech Zone, Dalian 116024, P.R. China
S
* Supporting Information
ABSTRACT: Identifying cancer cells and quantifying cancer-
related events in particular organelles in a rapid and sensitive
fashion are important for early diagnosis and for studies on
pathology and therapeutics of cancers. Herein a smart “off−
on” cyclooxygenase-2-specific fluorescence probe (ANQ-IMC-
6), able to report the presence of cancer cells and to image
Golgi-related events, has been designed and evaluated. Cyclooxygenase-2 (COX-2) has been used as imaging target in the probe
design, since this enzyme is a biomarker of virtually all cancer cell lines. In the free state in aqueous solution, ANQ-IMC-6 mainly
exists in a folded conformation where probe fluorescence is quenched through photoinduced electron transfer between the
fluorophore acenaphtho[1,2-b]quinoxaline (ANQ) and the recognition group, indomethacin (IMC). Fluorescence is turned on,
by restraining the photoinduced electron transfer, when ANQ-IMC-6 is forced to adopt the unfolded state following binding to
COX-2 in the Golgi apparatus of cancer cells. ANQ-IMC-6 provides high signal-to-background staining and has been successfully
used to rapidly differentiate cancer cells from normal cells when using flow cytometry and one- and two-photon fluorescence
microscopic imaging. Furthermore, ANQ-IMC-6 may be able to visualize dynamic changes of the Golgi apparatus during cancer
cell apoptosis, with possible application to early diagnosis.
accumulating in the Golgi apparatus.11 Consequently, if the
COX-2 in the Golgi apparatus of cancer cells could be
visualized, then it might be possible to assess the efficacy of
INTRODUCTION
■
Cancer is one of the major causes of death worldwide and in
2008 resulted in 7.6 million deaths (13% of the total).1,2 Early
treatment protocols and the cellular impact of pharmaceutical
diagnosis of cancer is particularly important for reducing cancer
targets, perhaps even permitting personalized oncology.11
mortality.2 In the past decade, molecular fluorescence
microscopy imaging has come to offer opportunities for the
It is in this context that we report a novel “off−on” COX-2-
specific fluorescent probe, ANQ-IMC-6 (Scheme 1), for rapid
identification of cancer cells by imaging. This probe localizes in
the Golgi apparatus and uses COX-2 as an imaging target. In
early diagnosis of cancer due to its high selectivity, high
resolution, and noninvasive capabilities.3,4 In particular,
molecular probes whose fluorescent signals are generated
aqueous buffer solution, ANQ-IMC-6 is present in the folded
(off−on probes) by enzymic biomarkers of cancers hold great
state. This results in the quenching of fluorescence due to
potential for identification and enumeration of living cancer
cells.5,6
Recently, Urano et al.,7 McCarley et al.,8 and Marnett et
Scheme 1. Chemical Structures of ANQ, IMC, and ANQ-
IMC-6 (Folded and Unfolded)
al.9,10 have designed fluorescent probes using overexpressed
enzymes as imaging targets in the cancer cells. Thus γ-
glutamyltranspeptidase (GGT), quinone oxidoreductase iso-
zyme 1 (NQO1), and cyclooxygenase-2 (COX-2) have all been
used to distinguish cancer cells from normal ones. These
probes have widened our picture of possible molecular design
ideas for the visualization of cancer cells. Regrettably, however,
none of them could localize at and image single organelles of
cancer cells. This limits their application for the study on the
outcomes of therapy and for accurate and early diagnoses.
Consider the Golgi apparatus for instance. This organelle is a
key structure for transporting and secreting some important
proteins/enzymes which are overexpressed in cancer cells, for
example COX-2. It has been suggested that when the stress-
signaling threshold is exceeded, the Golgi apparatus automati-
cally initiates apoptotic processes, which results in COX-2
Received: June 7, 2013
© XXXX American Chemical Society
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photoinduced electron transfer (PET) between the
acenaphtho[1,2-b]quinoxaline (ANQ, a fluorophore) and
indomethacin (IMC, an inhibitor of COX-2). However intense
fluorescence results from restraining PET following binding of
ANQ-IMC-6 to COX-2 in the Golgi apparatus. The probe
exhibits a large Stokes shift (90 nm) due to the push−pull
internal charge-transfer mechanism associated with the ANQ
scaffold. In this way the probe’s characteristics allow for the
rapid, selective, and sensitive identification of cancer cells. As
fluorescence intensity increase relates to the content of COX-2,
ANQ-IMC-6 may be of use for studying early cancer related
and dynamic changes of the Golgi apparatus during cancer cells
apoptosis, possibly facilitating early diagnoses.
RESULTS AND DISCUSSION
■
Design Strategy of Probe. For selective imaging of the
Golgi apparatus of cancer cells, COX-2 was chosen as the
imaging target because it is overexpressed in virtually all cancer
cell lines and accumulates significantly in the Golgi apparatus.9
ANQ was selected as the fluorophore, which results in ANQ-
IMC-6 displaying excellent fluorescence with two-photon
features, such as increased penetration depth, localized
excitation, and prolonged observation time.12−15 The hexanedi-
amine used as linker (Scheme 1) allows ANQ-IMC-6 to exist in
a folded conformation in aqueous solution. IMC, a good
inhibitor for COX-2,9,10 when linked to ANQ through
hexanediamine acts as the binding-site for COX-2. Application
of a molecular docking method (Figure S1a) indicates that
ANQ-IMC-6 binds to three amino acids of COX-2, i.e.,
Arg120, Tyr355, and Glu522, which is consistent with the
known interaction of IMC with COX-2.9,10
Figure 1. (a) Fluorescence emission spectra of ANQ-IMC-6 (2.0 μM)
in the absence and presence of COX-2 (0.50 μg/mL) in buffer at 25
°C. (b) Changes in fluorescence with different biomolecules (0.050
μg/mL) in buffer: 1, control; 2, COX-2; 3, RNA; 4, DNA; 5,
triacylglycerol acylhydrolase; 6, lysozyme; 7, proteinase k; 8, histone;
9, collagen; 10, hemoglobin; 11, BSA; 12, β-amylase; 13, trypsin; and
14, chymotrypsin. (c) Emission spectra of ANQ-IMC-6 (2.0 μM)
upon the addition of increasing concentrations of COX-2 (0−1.0 μg/
mL.) in buffer. (d) The fluorescence of ANQ-IMC-6 (2.0 μM) related
to COX-2 concentration (0.050−0.72 μg/mL). Condition: 100 mM
Tris-HCl buffer (pH 8.0). The data are obtained from replicate
experiments (n = 5).
The detailed synthetic route and chemical structure of ANQ-
IMC-6 are described in Scheme S1. The structures of ANQ-
IMC-6 and intermediates were well characterized by 1H NMR,
13C NMR, and TOF-MS (see Supporting Information text and
Figure S1).
the ANQ fluorophore moiety to extend into the large
hydrophobic cavity of COX-2s’ homodimer. These results
were supported by a molecular docking method (Figure S1b−
d). As a result of the unfolding, PET between ANQ and the
IMC moieties does not occur, thus restoring fluorescence16
(Figure 2b).
Spectral Properties and Discussion of Mechanism.
The optical properties of ANQ-IMC-6 were determined in
Tris-HCl buffer (100 mM, pH 8.0). There was a significant
fluorescence “off−on” process of ANQ-IMC-6 when interact-
ing with COX-2. When excited at 457 nm (ε = 16100 M‑1
ANQ-IMC-6 did not exhibit observable fluorescence in the
presence of various biomolecules, such as RNA, DNA,
triacylglycerol acylhydrolase, BSA, and so on (Figure 1b).
Furthermore, IC50, 50% inhibiting concentration of ANQ-IMC-
6 for COX-2 was used to evaluate the binding capacity of
ANQ-IMC-6 for COX-2. The IC50 values of ANQ-IMC-6 and
IMC for COX-2 were 0.73 and 0.75 μM, respectively,
indicating that the binding affinity of ANQ-IMC-6 for COX-
2 is as strong as that of IMC. Upon gradual addition of COX-2
to a solution of ANQ-IMC-6, the fluorescence intensity
increased linearly with the COX-2 concentration over the
range 0.12−0.72 μg/mL (Figure 1c,d). The detection limit for
COX-2 was determined as 0.11 μg/mL (3σ/k), higher than the
minimum expression of COX-2 (0.085 μg/mL) in cancer cells
(Figure 1d),17−19 demonstrating that ANQ-IMC-6 could be
used as a potential visual tool to selectively label cancer cells.
Verification for the Two-Photon Excitation Process of
ANQ-IMC-6. That ANQ-IMC-6 can undergo a two-photon
excitation process was confirmed by a power dependence
experiment. From the log−log plot of the emission intensity
against incident power (Figure 3a), a linear regression with a
slope of 2.06 was obtained, which indicates an obvious two-
photon excitation process. As shown in Figure 3b, the activated
two-photon action cross section (Φδ)max of ANQ-IMC-6 is 118
GM (1 GM = 10−50 cm4 s/photon) when excited at 800 nm in
the presence of COX-2 (0.50 μg/mL), and there was a 25-fold
cm‑1), ANQ-IMC-6 (2.0 μM) showed very weak fluorescence
Free
at 547 nm with ΦF
0.013. Upon binding to COX-2, it
COX‑2
showed a 28-fold enhancement in fluorescence with ΦF
0.39 (Figure 1a). To elucidate the mechanism of the
fluorescence “off−on” process, two conformations of ANQ-
IMC-6 (unfolded and folded, Scheme 1) were optimized, and
their frontier molecular orbital (FMO) energies were calculated
in water using Gaussian 09 (DFT/TDDFT in B3LYP/6-31G
level). The calculated results show that in water the energy of
the folded conformation is 3.0 kcal/mol lower than that of the
unfolded conformation. The distance between two parallel
planes of ANQ and IMC is about 0.36 nm. In this folded state,
there is PET between ANQ and IMC. The oscillator strength
from HOMO (S0) to LUMO (S1) is only 0.003, which means
that electron transition from HOMO (S0) to LUMO (S1) is
prohibited. Accordingly, the electronic transition from the
excited state to the ground state cannot happen, and the
fluorescence is quenched (Figure 2a). In the presence of COX-
2, the binding of the IMC moiety to COX-2 could force the
probe to adopt the unfolded conformation, because the
hydrophobic cavity of COX-2 can only hold the IMC moiety.
The hexanediamine linker is however long enough to enable
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Selectively Imaging Living Cancer Cells. Before study-
ing screening for living cancer cells by ANQ-IMC-6, enzyme
linked immunosorbent assay (ELISA) was used to determine
the amount of COX-2 in different cell lines. The data (Figure
4i) show that COX-2 was highly expressed in two cancer cell
Figure 4. Living cells stained with ANQ-IMC-6 (5.0 μM). (a, c, e, g)
white light images and (b, d, f, h) fluorescence images. Excitation
wavelength = 800 nm; scan range = 530−570 nm; and scale bar, 2.0
μm. (i) Quantification of COX-2 by ELISA (n = 5, p < 0.05). (j)
FCM: 1, Hela cells; 2, MCF-7 cells; 3, COS-7 cells; 4, HEK 293 cells.
(k) Response time of ANQ-IMC-6 in MCF-7 cells. Images are
representative of replicate experiments (n = 5).
Figure 2. Structural optimization and FMO of ANQ-IMC-6 (a) folded
and (b) unfolded calculated with time-dependent DFT using Gaussian
09.
lines (MCF-7 and Hela) but minimally expressed in two
normal cell lines (COS-7 and HEK 293). Following this assay,
the two cancer cell lines and two normal cell lines were
incubated with ANQ-IMC-6. Upon excitation at 800 nm
(TPM), both cancer cell lines showed strong and stable
fluorescence after incubation for 30 min (Figure 4b and 4d),
whereas both normal cell lines showed (Figure 4f and 4h)
negligible fluorescence even after prolonged incubation (120
min). Similar results were obtained following excitation at 488
nm (OPM), see Figure S2. The fluorescent microscopy imaging
results thus paralleled those seen with ELISA. All the results
indicate that ANQ-IMC-6 can distinguish cancer cells from
normal cells by labeling overexpressed COX-2 in cancer cells.
Subsequently, flow cytometry (FCM) was used to evaluate
the selectivity of ANQ-IMC-6 for cancer cells on large cell
populations. Histograms (Figure 4j) demonstrate that ANQ-
IMC-6 only stains cancer cells. Therefore, ANQ-IMC-6 shows
good applicability for the sorting of cancer cells in FCM.
Figure 3. (a) The log−log plot of emission intensity against incident
power. The linear regression equation is log δ = 2.06 log w − 0.58(R =
0.999), where δ is the two-photon cross section, w the incident power,
and R the correlation coefficient. (b) Two-photon action spectra of
ANQ-IMC-6 (2.0 μM) in the absence (black curve) and presence (red
curve) of COX-2 (0.50 μg/mL) in buffer at 25 °C. Buffer: 100 mM
Tris-HCl buffer (pH 8.0). Data are from replicate experiments (n = 5).
enhancement in the value (Φδ)max compared with that seen in
the absence of COX-2. These significant two-photon properties
make ANQ-IMC-6 suitable for two-photon microscopy
imaging applications in living specimens.20,21
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Furthermore, Figure 4k shows that ANQ-IMC-6 strongly
stained cancer cells within 0.5 h and that fluorescence intensity
remained almost unchanged in cells for at least 6 h. Therefore,
ANQ-IMC-6 is suitable for long-term observations in living
cancer cells.
Selective 3D Depth Imaging of Living Cancer Tissues.
To further assess ANQ-IMC-6 as a two-photon fluorescent
probe for bioimaging applications, we investigated the
applicability of this probe for tissue depth imaging. After
incubation of liver tissue slices and sarcoma 180 tissue slices
with 30 μM ANQ-IMC-6 for 30 min at 37 °C, TPM images
were obtained in the same region from 0 to 650 μm depth
excited at 800 nm. Significantly, the sarcoma 180 tissue slices
could be clearly visualized by green fluorescence at 50−550 μm
depth (Figures 5b,c and S3). Only negligible fluorescence was
observed in normal liver tissue slices.
Figure 6. Fluorescence images of ANQ-IMC-6 (5.0 μM) and
BODIPY TR C5-ceramide (5.0 μM) in HeLa cells. (a, b) Stained
with ANQ-IMC-6: (a) excitation wavelength = 800 nm, scan range =
530−570 nm; and (b) excitation wavelength = 488 nm, scan range =
530−570 nm. (c) Stained with BODIPY TR C5-ceramide, excitation
wavelength = 543 nm, scan range = 600−640 nm. (d) Merged image
of b and c. (e) Intensity correlation plot of stain ANQ-IMC-6 and
BODIPY TR C5-ceramide. Scale bar, 3.0 μm. Images and data are
representative of replicate experiments (n = 5).
μM) for 24 h. The two experiments indicate that ANQ-IMC-6
possesses excellent photostability and very low cell toxicity.
Imaging Morphological Changes of the Golgi
Apparatus during the Cancers Cells Apoptosis. ANQ-
IMC-6 was used to visualize the dynamic morphological
changes of the Golgi apparatus during the cancers cells
apoptosis. As shown in Figure 7, ANQ-IMC-6 stained the
Golgi apparatus of MCF-7 cells with green fluorescence (Figure
7a−c). When further incubated with an apoptosis inducer, tea
polyphenols, for 6 h,25 the Golgi apparatus was fragmented and
scattered near the nucleus (Figure 7d−f). The collapse is more
extensive after 24 h (Figure 7g−i). The dynamic changes of
cancer cells’ Golgi apparatus observed during cancer cell
apoptosis agree well with previous results obtained by scanning
electron microscope (SEM),26 providing a new mode of
investigating diagnostically relevant changes in early stage
cancer processes.
Figure 5. Living tissue slices stained with ANQ-IMC-6 (30 μM). (a,
d) white light images and (b, c, e) fluorescence images. Excitation
wavelength = 800 nm; scan range = 530−570 nm; and scale bar, 5 μm.
Images are representative of replicate experiments (n = 5).
Specific Localization of Golgi Apparatus of Cancer
Cells. A well-known fluorescent probe for the Golgi apparatus,
BODIPY TR C5-ceramide, was used to co-stain Hela cells with
ANQ-IMC-6 to determine its subcellular distribution. The co-
localization coefficient was evaluated by Pearson’s correlation
factor.22 Both the one photon fluorescence (OPM) (Figure 6a)
and TPM (Figure 6b) images stained by ANQ-IMC-6 match
very well with those stained by BODIPY TR C5-ceramide
(Figure 6c). The Pearson’s correlation factor is 0.97 (Figure
6e), indicating that ANQ-IMC-6 accumulates preferentially in
the Golgi apparatus of this cancer cell line. The similar results
(see Figure S4) were obtained in other cancer cell lines, such as
Saos-2 (bone cancer), MDA-MB-468 (breast cancer), MKN-
45(gastric cancer), Calu-3 (lung cancer), and HepG2 (liver
cancer).
Furthermore, the log P value of ANQ-IMC-6 (log P = 4.6)
was estimated by using the Hansch and Leo procedure.23 In
QSAR model, log P = 3−5 should be required for retaining in
Golgi.24 So ANQ-IMC-6 could enter into Golgi where it binds
to COX-2.
The fluorescence intensity of ANQ-IMC-6 remained >95%
in solutions irradiated with an iodine-tungsten lamp for 7 h
(Figure S5a) and >97% in cells after laser irradiation for 4 h
(Figure S5b). MTT assay (Figure S6) revealed that 92% of
HeLa cells survived after incubation with ANQ-IMC-6 (6.0
CONCLUDING REMARKS
■
We report a PET-quenched molecular probe (ANQ-IMC-6), a
fluorogenic derivative of a COX-2 inhibitor, whose fluorescent
signal is selectively and quickly generated by interaction with
COX-2 accumulating in the Golgi apparatus of cancer cells. The
“off−on” fluorescence enhancement results from restraining of
PET following ANQ-IMC-6 binding to COX-2 in the Golgi
apparatus of cancer cell lines. The push−pull charge-transfer
structure of ANQ-IMC-6 provides significant two-photon
properties permitting the selective identification of and
screening for cancer cells. As a result, the probe permits
rapid, highly selective and sensitive identification and
enumeration of cancer cells in FCM and under traditional
microscopy conditions via the imaging of Golgi apparatus of
live cancer cells by normal and two-photon fluorescence
microscopy. The two-photon system should be suitable for in
vivo and in vitro thick-specimen imaging (50−550 μm), where
the efficacy of treatment protocols and the cellular impact of
pharmaceutical targets can be evaluated for personalized
oncology. Moreover, ANQ-IMC-6 also visualized dynamic
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were calculated with time-dependent density functional theory (TD-
DFT)30,31 at the B3LYP/6-31G ** level.
Absorption and Fluorescence Quantum Yield Measure-
ments. Absorption spectra were measured on a Lamda LS35
spectrophotometer. Fluorescence spectra were obtained with a FP-
6500 spectrophotometer (Jasco, Japan). The relative fluorescence
quantum yields were determined using Rhodamine B (ΦF = 0.97 in
methanol) as a standard32 and calculated by the following equation:
Φ = Φ(F /F)(As/Ax)(λexs/λexx)(nx/ns)2
x
s
x
s
where Φ represents quantum yield; F is the integrated area under the
corrected emission spectrum; A is absorbance at the excitation
wavelength; λex is the excitation wavelength; n is the refractive index of
the solution (because of the low concentrations of the solutions,
10−7−10−8 mol/L, the refractive indices of the solutions were replaced
with those of the solvents); and the subscripts x and s refer to the
unknown and the standard, respectively.
Measurement of Two-Photon Cross Section. The two-photon
cross section (δ) was determined by using the femtosecond (fs)
fluorescence measurement technique as described in the literature.33
ANQ-IMC-6 was dissolved in different solution at a concentration of
5.0 × 10−4 M, and then the two-photon induced fluorescence intensity
was measured at 690−900 nm by using fluorescein (8.0 × 10−5 M, pH
= 11.0) as the reference, whose two-photon properties of this dye are
well characterized in the literature.34 The intensities of the two-photon
induced fluorescence spectra of the reference and sample resulting
from the same excitation wavelength were determined. The TPA cross
section was calculated by using equation:
Figure 7. Changes in Golgi apparatus morphology of cancer cells
during apoptosis. (a, b, d, e, g, h) Fluorescence images of MCF-7 cells
stained with ANQ-IMC-6 (5.0 μM). The concentration of tea
polyphenols was 100 μM. Excitation wavelength = 800 nm, and scan
range = 530−570 nm. (b, e, h) Amplifying images of a, d, and g. (c, f,
i) Merged images of white light and fluorescence images. The
incubation times of a, b, and c are 0 h, that of d, e, and f are 6 h, and
that of g, h, and i are 24 h. Scale bar: 0.5 μm. Images are representative
of replicate experiments (n = 5).
δ = δr(SsΦφcr)/(SrΦφcs)
r
s
s
r
where the subscripts s and r stand for the sample and reference
molecules. The intensity of the signal collected by a CCD detector was
denoted as S. Φ is the fluorescence quantum yield, and φ is the overall
fluorescence collection efficiency of the experimental apparatus. The
number density of the molecules in solution was denoted as c. δr is the
TPA cross section of the reference molecule. The measurements were
performed as above while stirring the mixture with a small magnetic
bar at 25 0.5 °C. The data were obtained from replicate experiments
(n = 5).
The figure is two-photon spectra, which was formed via the
activated absorption cross-section (Φδ) as y-axis and corresponding
excitation wavelengths as x-axis. The two-photon spectra can show
that the dyes have the highest two-photon absorption efficiency when
excited at which wavelength.
Cell Culture and Staining with ANQ-IMC-6. HeLa, MCF-7,
HEK293, COS-7, Saos-2, MDA-MB-468, MKN-45, Calu-3, and
HepG2 cells were purchased from Institute of Basic Medical Sciences
(IBMS) of the Chinese Academy of Medical Sciences. Cells were
cultured for 3 days in phenol red-free Dulbecco’s modified eagle’s
medium (DMEM, WelGene) supplemented with penicillin/strepto-
mycin and 10% fetal bovine serum (FBS; Gibco) in a CO2 incubator at
37 °C. One day before imaging, cells were seeded into a glass
bottomed dish (MatTek, 35 mm dish with 20 mm well) and were
incubated at 37 °C in a humidified incubator containing 5 wt % /vol
CO2. Before imaging, the live cells were incubated with 5.0 μM ANQ-
IMC-6 for 30 min at 37 °C under 5 wt % /vol CO2 and then washed
with phosphate-buffered saline (PBS) three times.
Preparation of Tissue Slices and Staining with ANQ-IMC-6.
Tumor tissue slices were prepared from nude mice with S180 sarcoma.
Normal tissue slices were prepared from livers of nude mice. The slices
were cut at 700 μm using a vibrating-blade microtome in artificial
cerebrospinal fluid (ACSF; 124 mM NaCl, 3 mM KCl, 26 mM
NaHCO3, 1.25 mM NaH2PO4, 10 mM D-glucose, 2.4 mM CaCl2, and
1.3 mM MgSO4). Slices were incubated with ANQ-IMC-6 (30 μM) in
ACSF bubbled with 95% O2 and 5% CO2 for 30 min at 37 °C and
then washed three times with ACSF and transferred to a glass
bottomed dish (MatTek, 35 mm dish with 20 mm well).
morphological changes of the Golgi apparatus during cancer
cell apoptosis. We anticipate that ANQ-IMC-6 could serve as a
practical tool for the early diagnosis of cancers.
EXPERIMENTAL SECTION
■
COX-2-Targeted Fluorescent Agent Preparation. The prep-
aration procedures of ANQ-IMC-6 and related intermediates are given
in the Supporting Information. All solvents and reagents used were
reagent grade. Silica gel (100−200 mesh) was used for flash column
chromatographic purification. BODIPY TR C5-ceramide was
purchased from Life Technologies Co. (U.S.A.). Tris base was from
Promega Co. (U.S.A.). COX-2 was obtained from Sigma Chemical Co.
(U.S.A.). Doubly purified water used in all experiments was prepared
using a Milli-Q system. The solutions of ANQ-IMC-6 were typically
prepared from 1.0 mM stock solutions in DMSO. Full details
regarding materials, synthesis of compounds, and their characterization
are described in the Supporting Information.
Mass spectral studies were carried out using a LC/Q-TOF mass
spectrometer. An auto sampler operated in-line with a Quantum triple
quadrupole instrument in ESI positive or negative ion mode. NMR
spectra were obtained using a Varian Inova 400 MHz (or a Bruker
Avance II 400 MHz) spectrometer. Experimental conditions included
2048 × 512 data matrix, 13 ppm sweep width, recycle delay of 1.5 and
4 scans per increment. The data were processed using squared sinebell
window function, symmetrized, and displayed in magnitude mode.
Quantum Calculations. All the quantum chemical calculations
were done with the Gaussian 09 suite.27 The parameter referred to the
work of Han.28 The geometry optimizations of the dyes were
performed using density functional theory (DFT)29 with Becke’s
three-parameter hybrid exchange function with Lee−Yang−Parr
gradient-corrected correlation functional (B3-LYP functional) and 6-
31G ** basis set. No constraints to bonds/angles/dihedral angles were
applied in the calculations, and all atoms were free to optimize. The
electronic transition energies and corresponding oscillator strengths
Two-Photon Fluorescence Imaging in the Cells and Tissues.
Two-photon fluorescence imaging of ANQ-IMC-6 in cells and tissues
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was obtained with a spectral confocal multiphoton microscope
(Olympus, FV1000) with a high-performance mode-locked titanium-
sapphire laser source (MaiTai, Spectra-Physics, U.S.A.). Numerical
aperture (NA) was 1.43 and 1.30, respectively. The intensity images of
ANQ-IMC-6 were recorded with the emission in the range of 530−
570 nm. To obtain images, internal PMTs were used to collect the
signals in 8 bit unsigned 1024 × 1024 pixels at 400 Hz scan speed.
Excitation wavelength: 800 nm. The images were obtained from
replicate experiments (n = 5).
Live Cell Dual Staining with BODIPY FL C5-Ceramide. ANQ-
IMC-6 and BODIPY FL C5-Ceramide stock solutions were added to
obtain 5.0 μM final concentrations. Cells were then incubated at 37 °C
under 5 wt % /vol CO2 for 30 min and then washed with phosphate-
buffered saline (PBS) three times. Fluorescence imaging was then
carried out with a spectral confocal one-photon microscope (Olympus,
FV1000), using a 100× objective lens. Excitation wavelength: 488 nm.
Green channel was collected at 530−570 nm, and red channel was
collected at 600−640 nm. The images were obtained from replicate
experiments (n = 5).
procedures described by Hansch and Leo. The predicted membrane
permeability and intracellular localizations for these species were
assessed by appropriate quantitative structure activity relations models
by methods described in the literature.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic protocol and characterization data for ANQ-IMC-6;
living cell imaging by OPM; 3D depth imaging in tissue;
photostability in solution and cell. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
fanjl@dlut.edu.cn; pengxj@dlut.edu.cn
■
Author Contributions
§These authors contributed equally.
3D Depth Imaging in Tissues. Depth fluorescence imaging of
ANQ-IMC-6 in tissues was obtained with a spectral confocal
multiphoton microscope (Olympus, FV1000) with a high-performance
mode-locked titanium-sapphire laser source (MaiTai, Spectra-Physics,
U.S.A.). The changes of fluorescence intensity with scan depth were
determined by spectral confocal multiphoton microscopy (Olympus,
FV1000) in the z-scan mode from 0 to 650 μm (step size = 3.0 μm).
Cytotoxicity. HeLa cells were prepared for cell viability studies in
96-well plates (1 × 105 cells per well that were incubated in 100 μL).
The cells were incubated for an additional 20 h with dyes ANQ-IMC-
6, NBD C6-Ceramide and BODIPY FLC5-Ceramide in different
concentrations. Subsequently, 100 μL of 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT, Sigma Chemical Co. U.S.A.)
was added into each well, followed by further incubation for 24 h at 37
°C. The DMEM was remove and DMSO (200 μL/well) added to
dissolve the reddish-blue crystals. Optical density (OD) was
determined by a microplate reader (Spectra Max M5, Molecular
Devices) at 570 nm with subtraction of the absorbance of the cell-free
blank volume at 630 nm. The results from the six individual
experiments were averaged. The relative cell viability (100%) was
calculated using the following equation:
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Science
Foundation of China (21136002, 20923006, and 21076032),
National Basic Research Program of China (2009CB724706
and 2013CB733702), National High Technology Research and
Development Program of China (863 Program,
2011AA02A105) and Ministry of Education (NCET-12-0080).
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