Ibuprofen-derived fluorescence inhibitor of COX-2 for breast cancer imaging, prevention and treatment

Article abstract of DOI:10.1016/j.dyepig.2021.109326

The integration of precise detection, long-term prevention and effective treatment are beneficial to monitor cancer bioactivity and prevent malignant metastasis for breast cancer patients. Herein, a novel fluorescence probe (IBLN) is designed and synthesized which contains a fluorophore (naphthalimide), a linker and a non-steroidal anti-inflammatory drug (ibuprofen). The specific optical imaging, prevention and treatment effects of breast cancer are relied on the introduction of a powerful cyclooxygenase-2-specific inhibitor, ibuprofen, which makes IBLN possible to successfully differentiate breast cancer cells from normal cells. In addition, fluorescent cellular imaging agent naphthalimide also has anticancer effects via inducing lysosomal membrane permeabilization (LMP). The multi-functional ibuprofen-derived fluorescence inhibitor IBLN realizes the imaging, prevention and treatment for breast cancer, which the selectivity and anticancer effects would be strengthened compared to the single naphthalimide or ibuprofen. Therefore, we envisage that the multi-function strategy in IBLN would be attractive way to explore potential reagent for fluorescence-visible breast cancer therapy in situ.

Full text of DOI:10.1016/j.dyepig.2021.109326

Dyes and Pigments 190 (2021) 109326
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: http://www.elsevier.com/locate/dyepig
Ibuprofen-derived fluorescence inhibitor of COX-2 for breast cancer
imaging, prevention and treatment
Wenxi Xiaa, Shuangzhe Zhanga, Yueqing Lia, Jiangli Fana, Bin Liub, Lei Wangb,
,
*
Xiaojun Penga
a State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, Dalian, 116024, PR China
b State Key Laboratory of Fine Chemicals, College of Materials Science and Engineering, Shenzhen University, Shenzhen, 518060, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The integration of precise detection, long-term prevention and effective treatment are beneficial to monitor
cancer bioactivity and prevent malignant metastasis for breast cancer patients. Herein, a novel fluorescence
probe (IBLN) is designed and synthesized which contains a fluorophore (naphthalimide), a linker and a non-
steroidal anti-inflammatory drug (ibuprofen). The specific optical imaging, prevention and treatment effects of
breast cancer are relied on the introduction of a powerful cyclooxygenase-2-specific inhibitor, ibuprofen, which
makes IBLN possible to successfully differentiate breast cancer cells from normal cells. In addition, fluorescent
cellular imaging agent naphthalimide also has anticancer effects via inducing lysosomal membrane per-
meabilization (LMP). The multi-functional ibuprofen-derived fluorescence inhibitor IBLN realizes the imaging,
prevention and treatment for breast cancer, which the selectivity and anticancer effects would be strengthened
compared to the single naphthalimide or ibuprofen. Therefore, we envisage that the multi-function strategy in
IBLN would be attractive way to explore potential reagent for fluorescence-visible breast cancer therapy in situ.
Breast cancer
Ibuprofen-derived fluorescence inhibitor
Cyclooxygenase-2
1. Introduction
early and precise detection of cancer [14–17]. Among these overex-
pressed enzymes, cyclooxygenase-2 (COX-2) as a rate-limiting enzyme
Breast cancer has become one of the most common invasive types
with greater than 130000 cases in women each year worldwide [1–3].
Long-term follow-up of randomized trials have shown that traditional
therapy for breast cancer such as radiotherapy, chemotherapy and
endocrine therapy still faced the challenges of easy metastasis, high
recurrence rate and the risk of heart disease [4–9]. In addition, the
predominant modalities for clinical detection and diagnostic of cancer
(including immunohistochemical analysis, positron emission tomogra-
phy, computed tomography and magnetic resonance imaging) [10,11]
are lack of sensitivity and specificity, which are difficult to achieve in
vivo real-time detection. Therefore, precise imaging and effective
treatment are the key to monitor cancer activity and prevent tumor
metastasis of cancer patients.
could catalyzes arachidonic acid to produce prostaglandin E2 (PGE2)
which interacts with the nuclear transcription factor (TCF)-4 to promote
the growth and proliferation of various cancers [18, 19]. COX-2 has been
verified to be up-regulated in various cancers, such as breast cancer, [20,
21] colon cancer [22,23], prostate cancer [24] and lung cancer [25], etc.
Therefore, the overexpressed COX-2 enzyme could be serve as a po-
tential detection and chemotherapy target for breast cancer [20, 26–28].
Ibuprofen as a COX-2 inhibitor as well as one of the non-steroidal
anti-inflammatory drugs (NSAIDs), could block endogenous prosta-
glandin synthesis through inhibition of COX-2 enzymatic activity and
thereby plays a role in cancer treatment and prevention [29–32]. Daily
intake of NSAIDs could produce risk reduction of 39% for breast. A few
studies have demonstrated that ibuprofen had a better anticancer effects
than aspirin, especially against breast and lung cancer [33]. Therefore,
the introduction of COX-2 inhibitor ibuprofen could specifically target
breast cancer cells, prevent the occurrence of cancers and inhibit cancer
proliferation [34] or invasion at the same time.
Optical imaging approaches provide the possibility of real-time,
noninvasive and high-resolution for in vivo cancer treatment [12,13].
Currently, fluorescence probes whose exhibit “off-on” fluorescent sig-
nals by utilizing specific overexpressed enzymes within cancer cells as
imaging targets have become a powerful biological strategy to achieve
In another aspect, the development of functional 1,8-naphthalimide
* Corresponding author.
E-mail address: pengxj@dlut.edu.cn (X. Peng).
https://doi.org/10.1016/j.dyepig.2021.109326
Received 12 December 2020; Received in revised form 11 March 2021; Accepted 12 March 2021
Available online 18 March 2021
0143-7208/© 2021 Elsevier Ltd. All rights reserved.

W. Xia et al.
Dyes and Pigments 190 (2021) 109326
derivatives as DNA targeting, anticancer and cell imaging agents is a
rapid developing area and has resulted in clinical applications for dual
therapeutic and fluorescence imaging [35]. There are two major anti-
cancer mechanisms: (1) naphthalimide structures can bind to DNA via
intercalation and inhibit topoisomerase II activity by interfering with
the breakage-rejoining step of the enzymatic cycle and stabilize the
enzyme-DNA cleavage complex [36]. (2) naphthalimide derivatives
could induce cancer cell apoptosis via lysosomal membrane per-
meabilization (LMP) [37], which releases cathepsins into the cytosol
and thereby triggers necrotic and apoptotic cell death pathways [38].
Among previous reports, even the most simple naphthalimide structures
also have prominent potential for clinical anticancer application [34].
Based on the above perspectives, we design and synthesis a fluo-
rescence imaging probe (IBLN) which contains a fluorophore (naph-
thalimide), a linker and a COX-2 kinase inhibitor (ibuprofen). The multi-
functional fluorescence probe IBLN realizes the combination with im-
aging, prevention and inhibition for breast cancer (Scheme 1), which the
highly selective differentiation and anticancer effects would be
strengthened compared to the signal naphthalimide or ibuprofen.
Interestingly, IBLN is successfully label and inhibit the growth of the
solid tumor in xenotransplantation model of nude mice.
166.08, 165.07, 164.47, 149.64, 147.84, 134.51, 131.84, 131.11,
129.80, 129.56, 128.79, 126.46, 126.30, 124.67, 123.26, 123.21,
123.01, 120.37, 118.54, 110.12, 104.29, 43.21, 39.30, 29.71, 29.69,
28.57, 26.80, 26.24, 26.03, 21.11, 14.12. HRMS (ESI): m/z calced for
C32H39N3O+3 [M+H]+ = 514.3064, found = 514.3065.
2.2. Fluorescence imaging of IBLN
MCF-7, HCT-116, COS-7 and H6C7 cells were seeded in glass-bottom
culture dishes and cultured for 24 h at 37 ◦C in a 5% CO2 humidified
incubator. Probe IBLN (5 μM) was added into the dish for incubation for
30 min before imaging on confocal laser scanning microscopy (CLSM)
with a 60 × objective oil lens.
2.3. Immunofluorescence assay
MCF-7 cells are seeded in a glass-bottom culture dishes and culture
for 24 h at 37 ◦C in a 5% CO2 humidified incubator. The cells are
incubated with IBLN (50 μM) for 8 h and wash twice with PBS. Then, the
cells are fixed with 4% poly formaldehyde solution (4% PFA) for 15 min
at room temperature, removed the PFA and washed twice with PBS. The
cells are added 0.1% Triton X-100, cultured for 5 min and washed twice
with PBS. Subsequently, the cells are blocked with 1% BSA for 30 min,
and then incubate with specific concentration COX-2 and C-myc anti-
body for 4 h at room temperature. After the incubation with antibody,
the cells are washed with PBS to remove the extra antibody, and then
added with Alexfluo647 lgG (H + L) for incubation 1 h. The immuno-
fluorescence imaging is observed under CLSM.
2. Experimental
2.1. Synthesis of IBLN
Target compound IBLN was obtained by the amidation reaction with
compound 3 (65 mg, 0.2 mmol) and ibuprofen (52 mg, 0.25 mmol) in
the presence of 1-(3 -dimethylaminopropyl)-3-ethylcarbodiimide hy-
drochloride (EDCI, 48 mg, 0.25 mmol) and 4-dimethylaminopyridine
(DMAP, 31 mg, 0.25 mmol). The mixture are dissolved in CH2Cl2 (20
mL) and stirred at r. t for 24 h under the N2 protection. The crude
product is purified by silica gel column chromatography with CH2Cl2:
CH3OH = 10: 1 and obtains the yellow solid. 1H NMR (500 MHz,
Chloroform-d) δ 8.58 (d, J = 7.3 Hz, 1H), 8.45 (d, J = 8.4 Hz, 1H), 8.30
(d, J = 8.4 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.19 (d, J = 7.6 Hz, 2H),
7.10 (d, J = 7.7 Hz, 2H), 6.69 (d, J = 8.4 Hz, 1H), 3.54 (s, 1H), 3.53 (s,
3H), 3.38–3.31 (m, 2H), 3.24 (s, 2H), 2.44 (d, J = 7.1 Hz, 2H), 1.83 (s,
1H), 1.73 (s, 2H), 1.53 (d, J = 7.1 Hz, 4H), 1.46 (s, 3H), 1.29 (s, 2H),
0.88 (d, J = 6.3 Hz, 6H). 13C NMR (126 MHz, Chloroform-d) δ 169.26,
2.4. Xenograft breast tumor inhibition efficacy and in vivo optical imaging
All the animal experiment is approved by Dalian Medical University
Animal Care and Use Committee. Subcutaneous breast cancer xenograft
is formed in 6 weeks female BALB/c nude mice through injecting 5 × 106
MCF-7 cells (100 μL) into each side of the axilla. After three days in-
jection, nude mice are divided into three groups and treated with PBS,
ibuprofen and IBLN (ibuprofen concentration: 40 mg/kg, IBLN con-
centration: 40 mg/kg) through gavage administration every day,
respectively. Body weight and tumor volume of each mice are measured.
The tumor volume is calculated as: tumor volume (mm3) = (1/2) ab2 (a
Scheme 1. Imaging and breast cancer treatment diagram of IBLN.
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W. Xia et al.
Dyes and Pigments 190 (2021) 109326
= tumor length, b = tumor width). IBLN (20
μ
L of 300
μ
M) is injected
3.3. Fluorescence imaging in living cells
into the subcutaneous tumor of mice and imaged by NightOWL II LB983
small animal in vivo imaging system with a 488 nm excitation.
In order to validate the selectivity of IBLN towards COX-2 enzyme in
vivo, the fluorescence images are recorded by confocal laser scanning
microscopy (CLSM). As shown in Fig. 3A, the time-dependent fluores-
cence experiments are first performed in MCF-7 cells. Upon the addition
3. Results and discussion
of 5 μM IBLN, the fluorescence intensity is gradually enhanced and in-
3.1. Synthesis and spectroscopic characteristics
creases to the maximum at 30 min, and then remains saturation for the
following 30 min (Fig. 3B). Next, two cancer cell lines (MCF-7 cells and
HCT-116 cells) and two normal cell lines (COS-7 cells and H6C7 cells)
are selected to investigate whether the overexpressed COX-2 enzyme
could be regarded as an effective biomarker for MCF-7 cells identifica-
tion. The concentration differences of COX-2 enzyme in various cell lines
are obtained by enzyme linked immunosorbent assay (ELISA, shown in
Fig. S5). Upon the excitation at 488 nm, obvious fluorescence signals are
The target fluorescence inhibitor IBLN is prepared and showed in
Scheme 2. The chemical structure of IBLN is characterized by HRMS, 1H
NMR and 13C NMR (Figs. S1–S3). The fundamental spectral and pho-
tophysical characteristics of IBLN are investigated and showed in
Table S1. As shown in Fig. 1 A, the UV–Vis spectrum of IBLN in different
solvents are demonstrated. The maximum absorption peak is 452 nm in
PBS buffer solution (0.01 M, pH 7.4). With the increase of solvent po-
larity, the emission wavelength of IBLN exhibits a bathochromic shift
(Fig. 1 B). As shown in Fig. 1C, UV–Vis spectra titration experiment of
IBLN indicates it exhibits a favorable solubility in PBS solution.
obtained after MCF-7 cells and HCT-116 cells incubated with 5 μM IBLN
for 30 min, whereas the two normal cell lines exhibit negligible fluo-
rescence signals (Fig. 3C and D).
To further demonstrate that IBLN is capable of binding with COX-2
enzyme, we pre-incubate a selective COX-2 inhibitor (Rofecoxib) with
Afterward, methyl thiazolytetrazolium (MTT) assay is carried out to
investigate the in vitro cytotoxicity of IBLN. MCF-7 cells are selected and
incubated with different concentrations of ibuprofen and IBLN for 24 h,
MCF-7 cells for 4 h with the concentration of 0
μ
M, 10
μM and 25 μM,
respectively. Then, MCF-7 cells are added with 5
μM IBLN and incubate
respectively. As shown in Fig. 1 D, the cell viability of 600 μM IBLN and
for 30 min before imaging. As shown in Fig. 3E and F, it is observed that
the fluorescence intensity decreases with the increase of Rofecoxib
concentrations. Overall, IBLN could serve as a prominent tool to
distinguish MCF-7 cells from normal cells by overexpressed COX-2
enzyme.
ibuprofen in MCF-7 cells are about 50% and 60%, and the corresponding
IC50 (half maximal inhibitory concentration) value of IBLN is 2.781 M,
M), indicating
μ
1.106-fold lower than that of ibuprofen (IC50 = 3.077
μ
that probe IBLN has stronger toxicity of cancer cells compared to
ibuprofen.
3.2. Binding mechanism
3.4. LMP induction
Docking calculations based on Discovery Studio 2.5 platform were
carried out to investigate the interaction between IBLN and COX-2
enzyme. The X-ray crystal structure of mouse COX-2 from PDB data-
base (PDB code 3LN1) is selected as interactions model. The binding site
is defined based on celecoxib. The ibuprofen part and celecoxib were
located in similar position in a narrow groove cavity and overlapped
well (Fig. 2A and B). As shown in Fig. 2C, the stretched structure of IBLN
could be docked well in the groove. In addition, the -CDOCKER Inter-
action energy of IBLN is ꢀ 69.7012 compared with that of ibuprofen is
ꢀ 36.7526, demonstrating that the structure of IBLN has stronger
binding at the COX-2 active site. These results exhibited strong consis-
tency with the MTT assay, suggesting that IBLN could more effectively
induce cell death.
Acridine orange (AO) tends to accumulate in normal lysosomes,
which is utilized to analyze the induction of LMP by the part naph-
thalimide [39]. LMP induces AO to migrate from lysosomes to cyto-
plasm, and the fluorescence signals changes from red to green.
Therefore, to evaluate the lysosomal disruption, red fluorescence signals
are collected and analysized by flow cytometry [40,41]. The LMP in-
duction is measured after MCF-7 cells incubated with six different
concentrations of IBLN. As shown in Fig. 4A, the amount of cells with
AO red fluorescence is decreased with the increasement of IBLN con-
centration. In addition, the percentage of AO red fluorescence is merely
12.51% compared with the control (87.97%), indicating significant
LMP. These findings demonstrated that LMP contributed to the cyto-
toxicity of MCF-7 cells using IBLN and the part naphthalimide could
Scheme 2. Synthetic route of IBLN. Reagents and Conditions: (a)methylamine, 86%, 100 ◦C, 2 h; (b) 2-methoxyethanol, 60%, 125 ◦C, 5.5 h; (c) EDCI, DMAP, DCM,
63%, rt, 24 h.
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Dyes and Pigments 190 (2021) 109326
Fig. 1. (A) The absorbance and (B) emission spectra of IBLN in different solvents (10
The cell viability of ibuprofen and IBLN in living MCF-7 cells for 24 h.
μ
M). (C) The linearity of IBLN absorbance intensity in PBS buffer solutions. (D)
Fig. 2. Docking results of IBLN bound to COX-2 enzyme. (A) The docking position of IBLN in COX-2. (B) The overlapped interaction structure between celecoxib and
IBLN. The carbon atoms of celecoxib were pink. (C) A stretched structure of IBLN locates in the narrow cavity of COX-2.
enhance the anticancer effect of IBLN.
surrounding the nucleus of MCF-7 cells. Meanwhile, the control has
insignificant fluorescence signal in MCF-7 cells, suggesting vivid mito-
chondrial outer membrane permeabilization (MOMP)-induced cell
apoptosis by IBLN.
As reported, LMP could induces cell death by cell apoptosis [38]. In
order to further investigate the potential mechanism of
apoptosis-induced cell death, the release of cytochrome c assays are
carried out by CLSM. The release of cytochrome c from mitochondria
into cytoplasm is an important process in the mitochondrial-dependent
apoptosis pathway, which causes cell apoptosis and activates apoptosis
gene including caspase-3 and caspase-9 [42, 43]. Antimouse Alex-
fluo647 antibody (red channel) is utilized to response the expression of
Subsequently, we further validate the cell apoptosis of IBLN using
Annexin V-FITC and propidium iodide (PI) indicator by flow cytometry.
MCF-7 cells are incubated with different concentrations of IBLN for 24
h. As shown in Fig. 4C, the amount of living cells are decreased with the
increase of IBLN concentration. The percentage of late apoptotic is
cytochrome c in MCF-7 cells after incubated with 150 and 300
μM IBLN.
81.9% after incubated with 600
the cell apoptosis.
μM IBLN, confirming IBLN could induce
As shown in Fig. 4B, the obvious red fluorescence is observed
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Dyes and Pigments 190 (2021) 109326
Fig. 3. (A) Time-dependent fluorescence imaging of IBLN (5
μ
M) in MCF-7 cells. (B) Quantitative analysis of fluorescence intensity is determined by randomly
selected 6 regions of interest (ROI) in MCF-7 cells. (C) Specific fluorescence imaging of MCF-7, HCT-116, COS-7 and H6C7 cells staining with IBLN (5 μM) for 30 min.
(D) Quantitative analysis of fluorescence intensity is determined by randomly selected 6 regions of interest (ROI) from each cells. (E) The effect of Rofecoxib on IBLN
staining. (F) Quantitative analysis of fluorescence intensity is determined by randomly selected 6 regions of interest (ROI) from each cells. Excitation wavelength =
488 nm, scan scope = 500–540 nm. Bar = 10
t-test.
μm. Date was assessed as mean ± SD *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 determined by Student’s
Fig. 4. (A) IBLN-induced LMP. Numbers reflect the percentage of AO red fluorescence. (B) The effect of IBLN on the redistribution of cytochrome C in MCF-7 cells by
immunofluorescence assay. MCF-7 cells are stained with mouse anti-cytochrome C and PE goat anti-mouse IgG. (C) The cell apoptotic of IBLN are analysized with
AV-PI staining. Excitation wavelength = 647 nm, scan scope = 620–650 nm. Bar = 20 μm.
3.5. Fluorescence imaging in tissue slices and in vivo
verify that IBLN is suitable for breast tumor labeling in vivo. The blood
circulation half-live of IBLN was determined to be 1.9 h (Fig. S6). In
addition, we further explore the fluorescence imaging of IBLN in tumor
and normal tissues. Tumor and normal tissues (heart, liver, spleen, lung
and kidney) are soaked and stained with IBLN for 30 min and imaging
by NightOWL II LB983 Imaging System. As is seen in Fig. 5B, the fluo-
rescence intensity of tumors is significantly higher than that of normal
tissues and the corresponding quantitative analysis is exhibited in
In order to investigate the capacity of IBLN to specifically identify
tumors, we establish a MCF-7 tumor-bearing xenotransplantation model
of nude mice. IBLN (20 μL of 500 μM) is subcutaneously injected into
two sites (the upper is tumor site; the lower is normal tissue site) of nude
mice. As shown in Fig. 5A, a significantly obvious fluorescence signals
are detected in the tumor site compared to normal tissue location, which
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Dyes and Pigments 190 (2021) 109326
Fig. 5. (A) Fluorescence image of MCF-7 tumor-bearing nude mice. (B) Ex vivo fluorescence imaging of organs (M1: heart, M2: liver, M3: spleen, M4: lung and M5:
kidney) and tumor (M6) from an MCF-7 tumor-bearing nude mouse. (C) Relative intensity of organs and tumor. (D) CLSM imaging of tumor and normal tissue are
treated with IBLN. Bar = 10 μm. (E) Layer by layer superposition of CLSM imaging of tumor slices. (F) 3D depth imaging of tumor tissue after IBLN staining.
Fig. 5C, demonstrating that IBLN could distinguish tumor from normal
tissues. In addition, the probe IBLN was intestinal metabolism during
the 90 min due to the fluorescence intensity in intestines was the
strongest (Fig. S7).
contributed to regulate cell proliferation and promote cell division. As
studies report that NSAIDs could decrease the proteins and mRNA levels
of C-myc in human cell lines [44–46]. Herein, we further investigate and
compare the effect of IBLN and ibuprofen on the expression of C-myc in
MCF-7 cells. As shown in Fig. 6A, the fluorescence intensity of MCF-7
cells incubated with IBLN and ibuprofen are greatly reduced. The
fluorescence signal of IBLN-treatment is lower than that of ibuprofen,
which reveals that IBLN could down-regulate the expression of down-
stream C-myc and more effective in inhibiting cell proliferation
compared to ibuprofen.
Then, the sections of tumor and normal tissues are prepared through
freezing-microtome and incubate with IBLN for 30 min before CLSM
imaging. As shown in Fig. 5D and E, the tumor slices exhibit remarkable
fluorescence signals collected from 500 to 540 nm than normal slices,
further suggesting probe IBLN could specifically target tumor tissues,
however, normal tissues are not stained. Furthermore, IBLN could reach
a staining depth of 90 μm by 3D depth imaging on CLSM (Fig. 5F).
3.7. Antitumor efficacy of IBLN in vivo
3.6. Immunofluorescence imaging
The treatment capacity of IBLN is evaluated through the xenograft
tumor inhibition experiment. In order to investigate the prevention of
IBLN for breast cancer, the mice are transplanted subcutaneous with
MCF-7 cells after intragastric administration of ibuprofen and IBLN for
three days. Subsequently, PBS, ibuprofen and IBLN continue giving to
mice by intragastric administration in three groups every other day until
the day 17. The body weight and tumor volume of each group are
measured and monitored during the treatment. As shown in Fig. 6B (a),
there is no significant difference in body weight between the treatment
group and control group, suggesting the low biotoxicity of ibuprofen and
IBLN in vivo. Tumor volume is considered as an useful indicator in
evaluating the anticancer capacity of drug. As shown in Fig. 6B (b),
tumor growth is effective suppressed during the administration of
ibuprofen and IBLN compared to the PBS treatment group. After the
administration stopped at day 17, both ibuprofen and IBLN groups
The above experiments demonstrate that IBLN has ability to inhibit
cell division. Subsequently, in order to further investigate the anticancer
mechanism of IBLN on breast cancer cells, immunofluorescence exper-
iments are carried out to validate the effect of IBLN on the expression of
COX-2 and C-myc enzyme within MCF-7 cells. Ibuprofen is selected as
the contrast. Firstly, we study the changes of COX-2 expression in MCF-
7 cells after incubation with 300 μM IBLN (green channel) for 18 h.
Antimouse Alexfluo647 antibody (red channel) is utilized as the same
time to response the expression of COX-2 enzyme within MCF-7 cells. As
shown in Fig. 6A, the fluorescence intensity of MCF-7 cells incubated
with IBLN is significantly reduced compared to the control, indicating
that the expression of COX-2 is down-regulated by IBLN treatment.
In addition, IBLN also regulates the expression of downstream
enzyme C-myc. C-myc as a downstream target gene of COX-2 is
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Dyes and Pigments 190 (2021) 109326
Fig. 6. (A) The effect of ibuprofen and IBLN on the expression of intracellular COX-2 and C-myc in MCF-7 cells by immunofluorescence assay. (B) Basic properties of
MCF-7 tumor-bearing nude mice during the treatment with ibuprofen and IBLN. (a) The changes of body weight. (b) The changes of tumor volume. (c) Tumor weight
comparison from different groups after treatment. (d) Representative tumors selected from different groups of tumor-bearing nude mice. (e) Photos of tumor-bearing
nude mice after administration. (f) Toxicity assessment of organs and tumor slices with H&E staining. Alexfluo: excitation wavelength = 488 nm, scan scope =
640–680 nm; Hoechst 33342: excitation wavelength = 405 nm, scan scope = 430–480 nm; IBLN: excitation wavelength = 488 nm, scan scope = 500–540 nm. Bar =
10 μm.
exhibit a trend of tumor growth and the final tumor volumes of these
two groups are about 90 mm3 and 30 mm3, respectively. The therapeutic
effect by IBLN and ibuprofen in MCF-7 tumor-bearing mice is present
from Fig. 6 B(e). Afterward, all of the mice are euthanized and the tu-
mors are extracted for direct observation comparison (Fig. 6Bc, d). These
findings demonstrate that IBLN has better prevention and anticancer
effects compared with ibuprofen in MCF-7 tumor-bearing mice model.
In addition, histological sections are stained with hematoxylin-eosin
(H&E). As shown in Fig. 6B (f), no significant histopathological abnor-
mality, degeneration, lesions and difference are observed between PBS
and IBLN treatment group, indicating low biotoxicity of IBLN to normal
tissues. All the above experiments reveal that IBLN not only inhibits
tumor growth but also shows advantageous biocompatibility. We sus-
pect that IBLN might be serve as an effective prevention and theranostic
agent.
imaging, prevention and treatment for breast cancer.
CRediT authorship contribution statement
Wenxi Xia: developed and produced the synthesis and testing of this
work represented. Shuangzhe Zhang: developed and produced the
synthesis and testing of this work represented. Yueqing Li: produced
Figure 2A of binding mechanism. Jiangli Fan: Writing – original draft.
Bin Liu: Writing – original draft. Lei Wang: Writing – original draft.
Xiaojun Peng: Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4. Conclusions
Acknowledgements
In conclusion, the multi-functional fluorescence probe IBLN which
contains a fluorophore (naphthalimide), a linker and a COX-2 kinase
inhibitor (ibuprofen) is designed and synthesized. It exhibits a promi-
nent fluorescence performance and low biotoxicity. The probe IBLN
could specifically target breast cancer cells by overexpressed COX-2
enzyme and release strong fluorescence, thus realizing the imaging of
tumor sites. Considering the anticancer ability, IBLN exhibits better
treatment effects via down-regulated the expression of COX-2 and C-
myc. Meanwhile, the introduction of fluorophore naphthalimide would
strengthen the treatment effects for breast cancer. More importantly, the
growth of tumor could be effective inhibited by IBLN administration in
advance, indicating that IBLN also has prevention function on cancer.
Overall, we expect that this kind of enzyme-targeting fluorescence
probes could be an effective way to seek multifunctional agents for
This work was supported by National Science Foundation of China
(project 22090011), NSFC-Liaoning United Fund U1908202.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dyepig.2021.109326.
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