Coupling the near-infrared fluorescent dye IR-780 with cabazitaxel makes renal cell carcinoma chemotherapy possible

Article abstract of DOI:10.1016/j.biopha.2019.109001

Renal cell carcinoma (RCC) has always been considered resistant to chemotherapy. IR-780 is a near-infrared fluorescent (NIRF) dye that can be efficiently taken up by RCC cells. Cabazitaxel is a cytotoxic drug that interferes with mitosis by acting on tubu

Full text of DOI:10.1016/j.biopha.2019.109001

Biomedicine&Pharmacotherapy116(2019)109001
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Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Coupling the near-infrared fluorescent dye IR-780 with cabazitaxel makes
renal cell carcinoma chemotherapy possible
Yu Zhenga,1, Ting Lanb,1, Di Weia,1, Geng Zhanga, Guangdong Houa, Jiarui Yuanc, Fei Yana,
Fuli Wanga, Ping Menga, Xiaojian Yanga, Guo Chend, Zheng Zhua, Zifan Lud, Wei Heb,⁎
,
Jianlin Yuana,⁎
a Department of Urology, Xijing Hospital, Fourth Military Medical University, Xi’an, 710032, Shaanxi Province, China
b Department of Chemistry, School of Pharmacy, Fourth Military Medical University, Xi’an, 710032, Shaanxi Province, China
c St. George’s University School of Medicine, West Indies, Grenada
d State Key Laboratory of Cancer Biology, Department of Pharmacogenomics, Fourth Military Medical University, Xi'an, 710032, Shaanxi Province, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Renal cell carcinoma (RCC) has always been considered resistant to chemotherapy. IR-780 is a near-infrared
fluorescent (NIRF) dye that can be efficiently taken up by RCC cells. Cabazitaxel is a cytotoxic drug that in-
terferes with mitosis by acting on tubulin. We chemically fused IR-780 and cabazitaxel into a new drug, Caba-
780, which is expected to increase the sensitivity of RCC to chemotherapy. Infrared spectrum, nuclear magnetic
resonance spectra, high-resolution mass spectra, and IR spectra were used for detecting structural character-
ization of the new synthetic drug Caba-780. The RCC cells lines ACHN and 786-O, as well as the non-cancerous
human embryonic kidney cell line HEK293, were used to assess the cytotoxicity and tumor-efficient uptake of
Caba-780 in vitro. The xenograft tumor-bearing mice and C57 mice were used to estimate the tumor-efficient
imaging of Caba-780 as well as the safety and efficacy of its anti-tumor effects in vivo. The new synthetic drug
Caba-780 retains the NIRF properties of IR-780. In vitro, Caba-780 was efficiently absorbed by the RCC cell lines
ACHN and 786-O, and had an inhibitory effect on their growth, clonogenicity migration, and invasion. At the
same time, Caba-780 retained the anti-tumor effect of cabazitaxel, which can inhibit the growth of tumor cells
and promote apoptosis by inhibiting mitosis. In vivo experiments showed that Caba-780 can be taken up and
imaged in tumor tissue, whereby it inhibits tumor growth. The novel fused molecule Caba-780 has application
prospects in the diagnosis and treatment of RCC and makes RCC chemotherapy possible.
Renal cell carcinoma
Near-infrared fluorescent dye
Chemotherapy
Organic anion-transporting polypeptide
1. Introduction
early diagnosis of RCC very difficult. Although ultrasonography, com-
puted tomography, and magnetic resonance imaging can aid the early
Renal cell carcinoma (RCC) is one of the most common malignant
tumors in the urinary system. According to the American epidemiolo-
gical survey, there were approximately 73,820 new cases of RCC
worldwide in 2019, placing it 6th and 8th in incidence among the
malignant tumors in men and women, respectively [1]. Early diagnosis
and surgery are the best way to treat RCC. Unfortunately, the onset of
RCC is insidious and clinical symptoms are not obvious, which makes
diagnosis of RCC, small lesions are still difficult to detect [2]. IR-780 is
a heptamethylcyanine near-infrared fluorescent (NIRF) dye. Our re-
search group previously discovered that IR-780 can be efficiently taken
up by RCC cells through the organic anion-transporting polypeptide
(OATP) superfamily transporters, which enables the diagnosis of even
small lesions through tumor tissue-specific imaging. This method can
even be used to specifically label and detect circulating tumor cells in
Abbreviations: BSP, sulfobromophthalein sodium; CCK-8, Cell Counting Kit-8; DAPI, 4′,6-diamidino-2-phenyindole; DMSO, dimethyl sulfoxide; EST, estradiol; FBS,
Fetal bovine serum; FITC, Annexin V-fluorescein isothiocyanate; HIF, hypoxia-inducible factor; HRMS, high-resolution mass spectra; IC50, half-maximal inhibitory
concentration; MDR-1, multi-drug resistance gene 1; NIRF, near-infrared fluorescent; OATP, organic anion-transporting polypeptide; PBS, phosphate-buffered saline;
P-gp, p-glycoprotein; PI, propidium iodide; RCC, renal cell carcinoma; RIF, rifampin; SIN, sincalide; SPF, specific pathogen free; STAT1, signal transducer and
activator of transcription 1; UV, ultraviolet
⁎ Corresponding authors.
E-mail addresses: weihechem@fmmu.edu.cn (W. He), jianliny@fmmu.edu.cn (J. Yuan).
1 These authors have contributed equally to this work.
https://doi.org/10.1016/j.biopha.2019.109001
Received 14 March 2019; Received in revised form 12 May 2019; Accepted 14 May 2019
0753-3322/©2019TheAuthors.PublishedbyElsevierMassonSAS.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Y. Zheng, et al.
Biomedicine&Pharmacotherapy116(2019)109001
Fig. 1. Chemical synthesis and characterization of Caba-780. (A) The synthetic process of Caba-780. (B) Methanol solutions of each drug. (C) Fluorescence intensity
curves of different concentrations of Caba-780 in methanol. (D) Ultraviolet absorption intensity curves of Caba-780 solutions (20 μM) in different solvents. (E)
Ultraviolet absorption intensity curves of different concentrations of Caba-780 in methanol.
the blood [3].
chemically bonding IR-780 and chemotherapeutic drugs. The aim was
to consider the fact that IR-780 can be efficiently absorbed by RCC cells,
so that they can act as “Charon on the Acheron” and make che-
motherapeutic drugs more concentrated intracellularly. If the new
compound can stably accumulate intracellularly, it would have a dual
effect combining cytotoxicity with imaging and could significantly in-
crease the sensitivity of RCC to chemotherapeutic drugs, finally en-
abling the use of chemotherapy to treat this recalcitrant type of cancer.
Targeted therapeutics have brought a quantum leap to the treat-
ment of metastatic RCC. However, 20–25% of patients do not respond
to targeted therapies, having tumors that display so-called “initial re-
sistance”. Furthermore, some RCC patients may develop resistance after
a period of targeted therapy [4]. Therefore, a new treatment method is
urgently needed, and chemotherapy has been re-emphasized, even if
RCC has been considered resistant to chemotherapy [5], and clinical
chemotherapy-related studies have not yielded satisfactory results
[6–8]. The possible molecular mechanisms of RCC’s resistance to che-
motherapy drugs include the overexpression of ATP-dependent drug
efflux pumps [9], tubulin drug target changes [10], loss of the Von
Hippel-Lindau gene [11], suppression of Hdm-2-regulated P53 expres-
sion caused by uncontrolled hypoxia-inducible factor (HIF) 1-alpha and
HIF2-alpha [12], as well as the overexpression of signal transducer and
activator of transcription 1 (STAT1) [13] and bcl-2 [14]. The most
widely accepted argument is the first one. This theory holds that the
overexpression of multidrug resistance 1 (MDR-1) gene causes an in-
crease in the levels of p-glycoprotein (P-gp), a protein of the ATP-de-
pendent drug efflux pump family. This protein can pump chemother-
apeutic drugs from the intracellular space, bringing their intracellular
concentration below the cytotoxic level, thus making RCC resistant to
chemotherapy [9,15,16].
2. Materials and methods
2.1. Chemical reagents
IR-780 was purchased from Heowns Biological Technology Co., Ltd.
(Tianjin, China), cabazitaxel was purchased from Dalian Meilun
Biological Technology Co., Ltd. (Dalian, China), sulfobromophthalein
sodium (BSP) was purchased from Alfa Aesar Chemical Co., Ltd.
(Tianjin, China), rifampin (RIF) was purchased from TCI Development
Co., Ltd. (Shanghai, China), sincalide (SIN) and estradiol (EST) were
from Medchem Express (MCE, USA), MitoSPy Orange CMTMRos, Lyso
Tracker Green DND‑26 and 4′,6-diamidino-2-phenyindole (DAPI) were
from Cell Signaling Technology (CST, USA), Cell Counting Kit-8 (CCK-
8) was from Dojindo Molecular Technologies, Inc. (Tokyo, Japan),
RPMI-1640, DMEM, and Fetal bovine serum (FBS) were from Gibco
(Gaithersburg, MD, USA), crystal violet, trypsin, dimethyl sulfoxide
(DMSO) and paraformaldehyde were from Thermo-Fisher Scientific
From this perspective, achieving an effective intracellular con-
centration of a chemotherapeutic drug is the key to successful che-
motherapy of RCC. Therefore, we synthesized a new compound by
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Y. Zheng, et al.
Biomedicine&Pharmacotherapy116(2019)109001
(Waltham, MA, USA).
20 μM for 2 h. After washing twice with PBS, cells were incubated with
20 μM Caba-780 for 30 min. Finally, the cells were fixed, stained, and
observed as described in 2.4.1. The software Image J (Version 1.8.0 for
Windows, Rawak Software, Inc. Germany) was used to semi-quantita-
tively analyze the fluorescence intensity of each group to deduce the
blocking rate. The blocking rate of each blocker was calculated as: 1－
blocking fluorescence intensity/original fluorescence intensity without
blocker.
2.2. Cell line and cell culture
The RCC cells lines ACHN and 786-O, as well as the non-cancerous
human embryonic kidney cell line HEK293 were purchased from the
Shanghai cell bank (Shanghai, China). All cells were cultured in
medium with 10% FBS (RPMI-1640 for ACHN and 786-O, DMEM for
HEK293) and penicillin (100 IU/mL)/streptomycin (100 μg/mL) in a
humidity-controlled incubator with 5% carbon dioxide at a constant
temperature of 37 °C.
2.5. Anti-tumor effect of Caba-780 in vitro
2.5.1. Cytotoxicity assay
2.3. Synthesis and identification of Caba-780
The CCK-8 method was used to detect the cytotoxicity of different
concentrations of Caba-780 on the RCC cells and normal kidney cells.
ACHN, 786-O and HEK293 cells in the logarithmic growth phase were
inoculated into 96-well plates at a concentration of 1 × 104 /well and
cultured for 24 h. Different concentrations (0,1,2,3,4,5,6–20 μM) of
Caba-780, IR-780, and cabazitaxel were added, and the incubation
continued for another 24 h. Subsequently, 10% CCK-8 reagent was
added at 100 μL per well and incubated at 37 °C for 3 h, and the ab-
sorbance of each well at 450 nm was measured using a microplate
reader (Sunrise, TECAN, China). The relative cell viability was calcu-
lated by comparing the absorbance of the experimental group to that of
the blank control group, and expressed in percentage points. The half-
maximal inhibitory concentration (IC50) values were calculated by the
software GraphPad Prism 6 (Version 6.01 for Windows, GraphPad
Software, Inc., USA).
The synthesis route of the new drug Caba-780 is shown in Fig. 1A.
IR-780 (1) (200 mg, 0.3 mmol) and piperazine (103 mg, 1.2 mmol)
were dissolved in anhydrous DMF. The mixture was stirred at 85 °C for
8 h under N2 atmosphere, and then compound 2 (118 mg, 55%) was
purified by flash column chromatography (CH2Cl2:CH3OH = 6:1). Ca-
bazitaxel (3) (200 mg, 0.24 mmol) and succinic anhydride (144 mg,
1.44 mmol) were dissolved in anhydrous DCM, and then Et3N (0.40 mL,
2.88 mmol) was added. After 4 h of reaction, compound 4 (196 mg,
87.3%)
was
purified
by
column
chromatography
(CH2Cl2:CH3OH = 15:1). A solution of 4 (200 mg, 0.153 mmol) and 2
(109 mg, 0.153 mmol) in anhydrous DCM was stirred at 0 °C, and then
EDC−HCl (44 mg, 0.230 mmol) and HOAT (32 mg, 0.230 mmol) were
added and incubated for 8 h. A new compound, Caba-780 (5) (109 mg,
43.6%), was finally obtained by flash column chromatography
(CH2Cl2:CH3OH = 30:1), and the overall yield was 24%. Infrared
spectrum, nuclear magnetic resonance spectra (1H NMR and 13C NMR),
high-resolution mass spectra (HRMS) and IR spectra were used for
structural characterization. The fluorescence intensity of Caba-780 so-
lutions with different concentrations was measured using a Transient
Steady-state Fluorescence Spectrometer (Edinburgh FLS9; Edinburgh,
UK). Ultraviolet (UV) absorption spectra of Caba-780 at different con-
centrations and in different solvents were recorded using a UV-VIS-NIR
Spectrometer (PE Lambda950; Perkin Elmer, USA).
2.5.2. Plate colony-formation assay
To evaluate the ability of Caba-780 to inhibit the clonogenicity of
ACHN and 786-O cells, the plate colony-formation assay was im-
plemented. ACHN and 786-O cells in the logarithmic growth phase
were seeded into 6-well plates at a density of 500 cells per well and
cultured for 24 h, after which Caba-780, IR-780, and cabazitaxel were
added separately. The concentration of each drug was 1 μM, and the
treatment time was 24 h. A blank control group without drugs was also
included. Next, the cells were recovered in medium with 10% FBS after
drug withdrawal and left to grow for 14 d. Afterwards, the cells were
fixed with 4% paraformaldehyde for 30 min and stained with 0.1%
crystal violet for 1 h to observe the formation of colonies.
2.4. Uptake and subcellular localization of caba-780 in RCC cells
2.4.1. Uptake of caba-780
The RCC cell lines ACHN and 786-O as well as normal human em-
bryonic kidney cell line HEK293 cells were seeded into confocal dishes
(1 × 104 cells per dish) and cultured for 24 h. Cells were incubated with
Caba-780 at a concentration of 20 μM for 30 min. The cells were fixed
with 4% paraformaldehyde after being washed 2 times with phosphate-
buffered saline (PBS), followed by counterstaining of nuclei with 0.1%
DAPI for 10 min. The stained cells were rinsed twice with PBS, water-
sealed, and observed under a laser confocal microscope (Olympus
FV1000, Tokyo, Japan) with an excitation wavelength of 700 nm and
an emission wavelength of 780 nm.
2.5.3. Migration assay
The scratch migration assay was performed according to a pre-
viously published protocol with minor modifications [17]. ACHN and
786-O cells from the logarithmic growth stage were seeded into 6-well
plates at 1 × 105 cells per well and cultured for 24 h. Then, the cells
were starved for another 24 h in serum-free RPMI-1640 medium. Sub-
sequently, Caba-780, IR-780, and cabazitaxel were added at a con-
centration of 2 μM and incubated for 3 h. A blank control group was
mock-treated at the same time. After removing the drug, a fine needle
was used to scratch the cell layer along an imaginary diameter of each
plate. After replacing the cell culture medium with serum-free RPMI-
1640 medium for 12 h, cell migration was observed by optical micro-
scopy (Olympus DP-72, Japan).
2.4.2. Subcellular localization of Caba-780
The ACHN and 786-O cells were seeded into confocal dishes
(1 × 104 cells per dish) and cultured for 24 h. Cells were incubated with
20 μM of Caba-780 for 30 min, after which the cells were stained with
MitoSPy Orange CMTMRos and Lyso Tracker Green DND-26 for 30 min
and 60 min, respectively. The stained cells were fixed and the nuclei
counter-stained as described in 2.4.1. The excitation and emission
wavelengths of Mito and LysoTracker for confocal laser microscopy
were 490/516 and 554/576 nm, respectively.
2.5.4. Invasion assay
The invasion assay was performed according to a previously pub-
lished protocol with minor adjustments [18]. The matrigel matrix (BD
Biosciences, San Jose, CA, USA) was dissolved at 4 °C and diluted to
1:40 in RPMI-1640 medium. In total, 100 μL of the diluted matrigel
matrix was added per transwell chamber, and kept at 37 °C until it
solidified. ACHN and 786-O cells were inoculated in the upper cham-
bers at 1 × 105 cells per chamber, and cultured in serum-free RPMI-
1640 medium. Next, 500 μL of medium with 10% FBS were added to
each of the bottom chambers. Caba-780, IR-780, and cabazitaxel were
separately added at a concentration of 5 μM to the upper chambers, and
2.4.3. Inhibition of Caba-780 uptake
The ACHN and 786-O cells were cultured for 24 h as described in
2.4.1. Then, the 4 different OATP blockers BSP, RIF, SIN, and EST were
individually incubated with the two types of cells at a concentration of
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Y. Zheng, et al.
Biomedicine&Pharmacotherapy116(2019)109001
a normally cultured group was set as a control. After 24 h, the invading
cells below the sieve membrane were fixed with 4% paraformaldehyde
for 30 min, and stained with 0.1% crystal violet for 1 h. The number of
invading cells was observed and recorded under an optical microscope.
intraperitoneally injected once a week for 4 weeks. The injection dose
was 20 nmol/mouse. The control group was given an equal volume of
PBS. During the experiment, the dietary habits and activity of nude
mice were observed, and the size of the tumors in the nude mice was
recorded using a Vernier caliper (tumor volume = length × width 2/2).
Four weeks after the first injection, the animals were sacrificed and the
tumors were removed, fixed, and weighed. Further, HE staining and
immunohistochemistry were performed on the tumor tissues.
2.5.5. Apoptosis assay
ACHN and 786-O cells were seeded into 6-well plates (1 × 105 cells
per well) and cultured for 24 h. Caba-780, IR-780, and cabazitaxel at a
concentration of 10 μM were added into different wells and the cells
cultured for another 24 h. Normally cultured cells served as controls.
Cells were digested by trypsin, centrifuged, washed twice with PBS, and
resuspended in a mixed buffer solution at a density of 1 × 106/mL. An
aliquot containing 100 μL cell suspension (1 × 105 single cells) was
transferred to a 5 ml tissue culture tube. Subsequently, 5 μL Annexin V-
fluorescein isothiocyanate (FITC) and 5 μL propidium iodide (PI) were
added to the cell suspension and cultured in darkness at room tem-
perature for 15 min. Then, 400 μL 1 × combined buffer was added to
each tube, and the samples were analyzed by flow cytometry (FACS
Aria, BD, Biosciences, USA) within 1 h. The apoptosis rate was defined
as the percentage of Annexin-positive and PI-negative cells with respect
to the total number of cells.
2.8. Acute toxicity test
Twenty male C57 mice were randomly divided into 4 groups and
received intraperitoneal injections of Caba-780 once a week for 4
weeks. The injection doses were 100 nmol/mouse (5-fold of the ther-
apeutic dose), 200 nmol/ mouse (10-fold of the therapeutic dose) and
400 nmol/ mouse (20-fold of the therapeutic dose). The control group
received an equal volume of PBS. The dietary habits and physical ac-
tivity of the mice were observed, and the weight changes of mice in
each group were recorded. After 4 weeks of treatment, the mice were
sacrificed and their main organs were examined pathologically and
observed by HE staining.
2.5.6. Cell cycle assay
2.9. Statistical analysis
Cells were seeded and cultured as described in 2.5.5 and co-in-
cubated with Caba-780, IR-780, and cabazitaxel (10 μM) for 24 h.
Normally cultured cells were used as control group. Subsequently, the
cells were collected and fixed using absolute ethanol. The samples to be
tested were centrifuged for 5 min at 1000 rpm, stationary liquid was
discarded, centrifuged again for 5 min at 1000 rpm, and washed twice
with 2 ml PBS. The supernatant was discarded and the cells were sus-
pended in 400 μL PBS. The cells were then incubated at 25 °C for 30 min
with 50 μL PI and 50 μL RNase. Finally, the cells were analyzed by flow
cytometry and the percentage of cells in each cell cycle phase was re-
corded.
The statistical software SPSS (Version 21.0 for Windows, IBM Corp.,
USA) was used to evaluate the data. The data were presented as
means
standard deviation. Variance test (one-way ANOVA) was
used to compare the data of multiple groups, and the t-test was used to
compare the data between two groups. Differences were considered to
have statistical significance at P < 0.05.
3. Results
3.1. Synthesis and physicochemical properties of Caba-780
2.6. Caba-780 in vivo imaging assay
The final product Caba-780 was a blue solid powder, and its
structure was confirmed by 1H NMR, 13C NMR, HRMS and IR spectra
(Supplementary Fig.1). The corresponding data were as follows: 1H-
NMR(CDCl3,400 MHz): 1H NMR (400 MHz,) δ: 7.729 (d, J =13.6 Hz,
2 H), 7.361 (d, J =7.2 Hz, 5 H), 7.284 (s, 6 H), 7.168 (t, J =14.8 Hz
3 H), 7.015 (d, J =8 Hz, 2 H), 5.879 (d, J =13.2 Hz, 2 H), 3.961– 3.925
(m, 12 H), 3.711 (br, 3 H), 3.239– 3.172 (m, 11 H), 2.509 (t, J
=12.8 Hz, 6 H), 1.917– 1.855 (m, 14 H), 1.503 (s, 3 H), 1.484 (s, 8 H),
1.466 (s, 5 H), 1.270 (s, 10 H), 1.083 (t, J =14.8 Hz, 13 H), 0.909 –
0.853 (m, 7 H). 13C-NMR (CD3OD, 400 MHz): 207.642, 207.542,
176.131, 173.797, 172.214, 171.732, 171.614, 170.461, 167.62,
144.131, 143.227, 141.96, 141.478, 139.06, 136.062, 135.953,
134.631, 134.087, 131.353, 131.248, 129.966, 129.726, 129.427,
128.414, 126.987, 125.271, 123.331, 122.065, 111.346, 85.515,
83.761, 82.426, 81.984, 80.871, 79.643, 79.316, 79.017, 77.384,
76.528, 76.026, 73.183, 58.143, 57.462, 57.092, 56.27, 55.387,
54.967, 49.886, 48.006, 46.092, 44.637, 36.421, 33.058, 30.747,
30.029, 29.872, 29.311, 29.18, 28.764, 27.224, 26.146, 23.404,
22.968, 21.858, 21.55, 15.165, 15.036, 14.465, 11.791, 10.971, 9.302.
2.6.1. Establishment of tumor-bearing mice
All animal experiments were approved by the ethics committee of
the Fourth Military Medical University and carried out in accordance
with the EU Directive 2010/63/EU for animal experiments. All ex-
perimental animals were purchased from the Experimental Animal
Center of Fourth Military Medical University. The mice were reared in a
specific pathogen free environment with ad libitum access to food and
water with a 14 h/10 h day and night cycle, at a controlled temperature
(22
1 °C) and humidity (50
10%). The concentration of ACHN
cells in the logarithmic growth phase was adjusted to 1 × 107/mL and
mixed with matrigel at a ratio of 1:1. The ACHN cells were injected
subcutaneously into nude mice at 0.2 mL/mouse. Nude mice that de-
veloped tumors with a diameter > 5 mm were used for subsequent
experiments.
2.6.2. Caba-780 tumor-efficient imaging assay
Caba-780, IR-780, and cabazitaxel were separately injected in-
traperitoneally into tumor-bearing nude mice at 20 nmol/mouse. Each
group included 5 mice. At 24 h after the injection, the mice were an-
esthetized with pure oxygen containing 2% isoflurane at a flow rate of
1.5 L/min. Finally, the IVIS Lumina II imaging station (Caliper Life
Sciences, USA) was used to observe the uptake of Caba-780 and IR-780
by the tumor, as well as their tumor imaging properties, with an ex-
citation wavelength of 700 nm and an emission wavelength of 750 nm.
HRMS: measured value 1507.8000, theoretical value C89H112N5O16+
:
1507.7689. The new drug Caba-780 was soluble in lipophilic solvents.
The methanol solutions (20 μM) of Caba-780, IR-780 and cabazitaxel at
room temperature were blue, light green and colorless/transparent,
respectively (Fig. 1B). The Caba-780 methanol solution exhibited the
strongest fluorescence at 5, 10 and 20 μM (Fig. 1C). Interestingly, this is
also the most commonly used concentration range for biological effi-
cacy. However, the fluorescence intensity did not increase with in-
creasing concentration. Most organic fluorescent dyes show strong
fluorescence in solution, but their fluorescence intensity will decrease
or even disappear in highly concentrated solutions or solid state. This
phenomenon of fluorescence quenching is mainly caused by the
2.7. Assay for the in-vivo anti-tumor activity of Caba-780
Twenty tumor-bearing male nude mice were divided into 4 groups,
with 5 mice in each group. Caba-780, IR-780, and cabazitaxel were
4

Y. Zheng, et al.
Biomedicine&Pharmacotherapy116(2019)109001
Fig. 2. Efficient uptake, subcellular localization and uptake blockage of Caba-780 in renal cell carcinoma (RCC) cells. (A) Efficient uptake of Caba-780 by RCC
cells. Strong fluorescence intensity (red) can be seen in ACHN and 786-O cells, while only a weak fluorescence signal can be seen in normal human embryonic kidney
cells HEK-293 (magnification, 200×). DAPI was used to counterstain nuclei (blue). (B) Subcellular localization of Caba-780. The top row shows the ACHN cells,
and the bottom row the 786-O cells (magnification, 1800×). DAPI was used to counterstain nuclei (blue). The fluorescence signal of Caba-780 is red. The signals of
MitoSPy Orange CMTMRos (Mito Tracker) and Lyso Tracker Green DND‐26 (Lyso Tracker) are orange and green, respectively. In the merge map, the signals of Mito
Tracker and Lyso Tracker are coincided with Caba-780, indicating that Caba-780 is located in the mitochondria and lysosomes of the cells after absorption. (C)
Blockage of Caba-780 uptake. The fluorescence intensity of Caba-780 (red) in ACHN cells treated with four kinds of OATP blockers decreased to varying degrees.
BSP and RIF had stronger inhibitory effects, while SIN and EST were relatively weak. DAPI was used to counterstain nuclei (blue) (magnification, 200×). This
phenomenon confirms that Caba-780 is transported into the cells through the OATP family transporters.
formation of intermolecular aggregation, which is called aggregation-
induced quenching [19]. The UV absorption spectrum of Caba-780
solutions in different solvents showed that the methanol solution with
the best solubility has the strongest UV absorption intensity (Fig. 1D),
and the UV absorption intensity of the Caba-780 methanol solution
increased with increasing concentration (Fig. 1E).
degrees (Fig. 2C), which indicates that Caba-780 is transported into the
cells through the OATP superfamily transporters. The semi-quantitative
analysis of the fluorescence intensity of the image showed that the
blocking rates of BSP, RIF, EST, and SIN on ACHN uptake of Caba-780
were 49.12%, 59.79%, 44.19%, and 44.30%, respectively, and the
blocking rates in 786-O were 55.97%, 54.59%, 36.87%, and 29.59%,
respectively.
3.2. Caba-780 uptake by RCC mediated by OATP superfamily transporters
3.3. In-vitro anti-tumor activity of Caba-780
Our previous research confirmed that IR-780 can be taken up by
RCC cells, so we utilized the characteristics of IR-780, and hoped that
the synthesized Caba-780 can also be taken up by RCC cells. Studies
have shown that Caba-780 is efficiently taken up by RCC cell lines
ACHN and 786-O, whereas HEK293 cells practically import a small
quantity of the molecule (Fig. 2A). This kind of tumor-efficient uptake
indicates that Caba-780 may have application prospects in tumor
imaging and treatment. At the same time, we stained the cells with the
mitochondrial tracer MitoSPy Orange CMTMRos and the lysosome
tracer Lyso Tracker Green DND-26. The results showed that the region
of NIRF and the stained regions of mitochondria and lysosomes largely
overlapped, which confirmed that after ingestion by RCC cells Caba-
780 was mainly located in membranous organelles including mi-
tochondria and lysosomes (Fig. 2B). Finally, the 4 different OATP su-
perfamily blockers BSP, RIF, SIN, and EST, were incubated with RCC
cells, and were found to be able to block Caba-780 uptake to varying
3.3.1. Cytotoxicity assay
Cytotoxicity experiments showed that the killing effect of Caba-780
on ACHN and 786-O cells was superior to that of cabazitaxel and IR-
780, especially at low concentrations. The toxicity of Caba-780 toward
HEK293 cells was stronger than that of cabazitaxel (Fig. 3A). The IC50
of cabazitaxel for the three kinds of cells could not be calculated, in-
dicating that the concentration was out of the range of concentrations
used in our experiments. This also confirmed that RCC cells were re-
sistant to chemotherapeutic drugs. The IC50 values of Caba-780 for
ACHN and 786-O cells were 11.5 and 1.9 μM, respectively, while those
of IR-780 were 31.2 and 10.4 μM. Similarly, the IC50 values of Caba-
780 and IR-780 for HEK293 cells were 8.7 and 5.5 μM, respectively.
Thus, Caba-780 promisingly, exhibited an increased toxicity in cancer
cells, while its toxicity in normal cells was weaker than that of IR-780.
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Fig. 3. In vitro effect of Caba-780 on RCC cells and normal human embryonic kidney cells. (A) Cytotoxicity assay using the Cell Counting Kit-8 (CCK-8)
method. ACHN, 786-O, and HEK293 cells were incubated with different concentrations (0–20 μM) of Caba-780, IR-780, and cabazitaxel. The IC50 values of Caba-
780 for ACHN, 786-O, and HEK293 cells were 11.5 μM and 1.9 μM, 8.7 μM respectively, and the IC50 values of IR-780 for ACHN, 786-O, and HEK293 were 31.2 μM
and 10.4 μM, 5.5 μM, respectively. The IC50 of cabazitaxel could not be calculated for each group. Dose-dependent inhibition of cell growth by Caba-780 was found.
The inhibitory effect of Caba-780 on tumor growth was stronger than that of other drugs, and its toxicity to normal cells was weaker than that of IR-780. (B) Plate
colony-formation assay. IR-780 had little effect on the clonal growth of the two RCC cell lines (ACHN on the left and 786-O on the right), and the cabazitaxel group
had some clone formation, while there was almost no colony formation in the Caba-780 group.
3.3.2. Plate colony-formation assay
yielded
P
values 0.0004, < 0.0001, and < 0.0001, respectively
The results of the plate clonogenicity experiments showed that IR-
780 had little effect on the clonal growth of the two RCC cell lines
compared with the control group. While the cabazitaxel group showed
some remaining clonogenicity, there was almost no colony formation in
the Caba-780 group (Fig. 3B). These results suggest that Caba-780 has a
stronger inhibitory effect on the clonal growth of ACHN and 786-O cells
than cabazitaxel or IR-780.
(Fig. 4D). The results showed that Caba-780 inhibits the migration
ability of RCC cells more effectively than cabazitaxel or IR-780.
3.3.4. Invasion assay
The number of cells that invaded from the transwell chamber in the
Caba-780 group was significantly smaller than that in the cabazitaxel
group, IR-780 group or the control group (Fig. 4E). In ACHN cells, when
the effect of Caba-780 treatment was compared with that of cabazitaxel,
IR-780, and the control, the P values were 0.0194, < 0.0001 and <
0.0001, respectively. In 786-O cells, the same comparison yielded P
values < 0.0001 (Fig. 4F).
3.3.3. Migration assay
After 12 h, the migration distance of the two types of RCC cells in
the Caba-780 group was smaller than that in the cabazitaxel, IR-780,
and control groups (Fig. 4A and C). When the effect of Caba-780
treatment was compared to that of cabazitaxel, IR-780, and the control
group in ACHN cells, the P values were 0.0128, < 0.0001, and <
0.0001, respectively (Fig. 4B). In 786-O cells, the same comparison
3.3.5. Flow cytometry
The effect of each group of drugs on the apoptosis of RCC cells was
determined by flow cytometry (Fig. 5A and Supplementary Fig. 2A).
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Biomedicine&Pharmacotherapy116(2019)109001
Fig. 4. In vitro effect of Caba-780 on RCC cells. (A) Migration assay for ACHN cells. From left to right are the control, IR-780, cabazitaxel, and Caba-780 groups.
The first horizontal row is the spacing state of the initial cell scratches. The second horizontal row is the state in which the cell breaks through the scratch and
migrates to the middle space after 12 h. (B) Statistical analysis of the ACHN migration assay. The migration distance of cells in the Caba-780 group was less than
that in other groups. (C) Migration assay for 786-O cells. From left to right are the control, IR-780, cabazitaxel, and Caba-780 groups. The first horizontal row
indicates the initial cell scratches. The second horizontal row indicates the migration of cells through the scratch after 12 h. (D) Statistical analysis of the ACHN
migration assay. The migration distance of cells in the Caba-780 group was less than that in other groups. (E) Invasion assay for ACHN (above) and 786-O cells
(below). From left to right are the control, IR-780, cabazitaxel, and Caba-780 groups. In the two kinds of RCC cells, the number of invasive cells in the Caba-780
group was significantly less than that in other groups. (F) Statistical analysis of the migration assay for ACHN (above) and 786-O cells (below). Whether in
ACHN or 786-O, the number of invasive cells in the Caba-780 group was less than that in other groups. *P＜0.05, **P＜0.01.
Compared with cabazitaxel, IR-780, and the control group, Caba-780
significantly promoted apoptosis of ACHN and 786-O cells, with all P
values < 0.0001 (Fig. 5C Supplementary Fig. 2C). Similarly, cell cycle
analysis suggested that Caba-780 significantly prolonged the G2 phase
of ACHN and 786-O cells (Fig. 5B and Supplementary Fig.2B). Com-
pared with other groups, Caba-780 group had shorter G1 phase and
longer G2 phase. All differences were statistically significant at P
values < 0.0001 (Fig. 5C and Supplementary Fig.2C). Regarding the
differences in S phase of ACHN cells, compared with IR-780, cabazi-
taxel, and the control groups, the P values were 0.0364, 0.9576, and
0.007, respectively. The corresponding P values for 786-O cells were
0.0132, 00197, and 0.0014, respectively (Fig. 5C and Supplementary
Fig.2C). Due to the different proliferative capacity, Caba-780 did not
significantly inhibit the S phase of ACHN cells. These results indicated
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Biomedicine&Pharmacotherapy116(2019)109001
Fig. 5. Apoptosis and cell cycle assay for ACHN cells. (A) Apoptosis assay for ACHN cells. From left to right are the control, IR-780, cabazitaxel, and Caba-780
groups. The apoptosis rate was defined as the percentage of Annexin-positive and PI-negative cells with respect to the total number of cells. (B) Cell cycle assay for
ACHN cells. From left to right are the control, IR-780, cabazitaxel, and Caba-780 groups. The red peaks on the left side of each group represent the percentage of cells
in the G1 phase, the red peaks on the right represent the percentage of cells in the G2 phase, and the shaded bottoms represent the percentage of cells in the S phase.
(C) Statistical analysis of the apoptosis assay (left) and cell cycle assay (right) for ACHN. The apoptotic rate in the Caba-780 group was higher than that in the
other groups. Meanwhile, the proportion of G2 phase cells in the Caba-780 group was higher than that in other groups. *P＜0.05, **P＜0.01.
that Caba-780 inherited the anti-tumor properties of cabazitaxel, in-
creasing the number of cells at the G2 phase of the cell cycle, and in-
hibiting mitosis.
tumors, highlighting their extent (Fig. 6A); cabazitaxel does not allow
tumor imaging. Animal experiments were also performed to compare
the therapeutic effects of Caba-780, IR-780, and cabazitaxel. The
growth rate of tumors in the Caba-780 group was significantly lower
than that in the cabazitaxel group, IR-780 group or control group, and
the differences were highly significant, with P values of 0.0005, 0.0037
and 0.0069, respectively (Fig. 6B). After the mice were sacrificed, sig-
nificant differences were revealed in the volume and weight of tumor
tissues between the Caba-780 group and the cabazitaxel, IR-780, and
3.4. In-vivo uptake and antitumor effects of Caba-780
Twenty four h after intraperitoneal injection of Caba-780, IR-780, or
cabazitaxel in tumor-bearing nude mice, the small animal imaging
system showed that Caba-780 and IR-780 could aggregate in the
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Biomedicine&Pharmacotherapy116(2019)109001
Fig. 6. In vivo imaging and anti-tumor assay of Caba-780. (A) in vivo imaging assay. Caba-780 and IR-780 accumulated in the tumors highlighting their extents,
while cabazitaxel did not. (B) Tumor growth curves of xenograft, Caba-780 (blue), cabazitaxel (red), IR-780 (green), and control groups (black). The tumor
growth rate in Caba-780 group was slower than that in cabazitaxel, IR-780, and control groups. (C) Tumor size at day 28 in each group. Each horizontal row
represents a different group (n = 5). From top to bottom, are the control, Caba-780, IR-780, and cabazitaxel groups. The tumor volume of the Caba-780 group is
smaller than that in the other treatment groups and control group. (D) Statistical analysis of tumor weight at 28 days. The weight of tumors in the Caba-780 group
was less than that in the other treatment groups and control group. *P＜0.05, **P＜0.01.
control groups, with P values of 0.0022, 0.0136 and 0.0306, respec-
3.6. Acute toxicity test
tively (Fig. 6C and D).
There were no significant differences in diet, behavior and activity
of C57 mice in each group during drug administration. Moreover, there
was no difference in body weight between the groups (Fig. 8A). The P
values of the groups administered a 5-fold of the therapeutic dose
(100 nmol/mouse), 10-fold of the therapeutic dose (200 nmol/ mouse)
and 20-fold of the therapeutic dose (400 nmol/ mouse) were 0.0800,
0.0754 and 0.7220 compared with the control group, respectively.
After the mice were sacrificed, HE staining of the main organs (Fig. 8B)
showed no degeneration or necrosis. These results indicate that Caba-
780 has no obvious adverse effects on the metabolism and main organs
of mice at the administered concentrations.
3.5. Immunohistochemistry
HE staining showed that the tissues in each group had cells with
large volumes, round or polygonal shape, abundant cytoplasm, trans-
parent or granular shape, obvious cell atypia, more mitotic figures,
deep nuclear staining, and typical clear cell-like changes, which con-
firmed that the tumors in each group were indeed RCC (Fig. 7A). Im-
munohistochemical staining also showed that Ki-67 expression was of
the same intensity in each group of RCC tissues, indicating that each
tumor group had the same proliferative capacity (Fig. 7B). There were
significantly more TUNEL-positive cells in the RCC tissues of the Caba-
780 group compared to the other groups, which indicated that Caba-
780 increased apoptosis of tumor cells. Importantly, Caba-780 also
promoted apoptosis of RCCs in vivo (Fig. 7B). Additionally, the ex-
pression of Cyclin D1 in the Caba-780 group was significantly decreased
compared to that in the other groups, which indicated that Caba-780
interferes with the mitosis of cancer cells by inhibiting the transition to
the G1 phase of the cell cycle (Fig. 7B).
4. Discussion
RCC is a malignant tumor originating from renal tubular epithelial
cells. It is one of the most common malignant tumors in the urinary
system. Nephron-sparing surgery or radical nephrectomy can benefit
the survival of patients with localized RCC (T1-T2) without distant
metastasis [20]. However, due to the insidious nature of RCC with few
initial symptoms, approximately 15% of patients already have
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Biomedicine&Pharmacotherapy116(2019)109001
Fig. 7. HE staining and immunohistochemistry of tumors. (A) HE staining of tumors. HE staining confirmed that the tumors in each group were indeed RCCs
(magnification, 200×). (B) Immunohistochemistry of tumors. The expression of Ki-67 (green fluorescent signal) was the same in all groups, indicating the same
proliferative ability of the tumors (top). The number of TUNEL-positive cells (green fluorescent signal) in the Caba-780 group was significantly increased, which
indicated that the apoptosis of tumor cells in this group was increased (middle). The decrease in CyclinD1 (green fluorescent signal) expression in the Caba-780 group
indicates that the G1 phase subpopulation was reduced (bottom) (magnification, 200×).
Fig. 8. Acute toxicity test. (A) Body weight curve of mice. There were no statistically significant differences in body weight between the groups administered with
a 5-fold (blue), 10-fold (green), 20-fold (red) increased concentration of the therapeutic dose and control group (black). The P values of the groups administered a 5-
fold increase of the therapeutic dose (100 nmol/mouse), 10-fold (200 nmol/ mouse) and 20-fold of the therapeutic dose (400 nmol/ mouse) were 0.0800, 0.0754 and
0.7220 compared with the control group, respectively. (B) HE staining of major organs in mice. No visceral degeneration or necrosis was found in any of the
groups (magnification, 200×).
metastatic RCC at the time of diagnosis [1]. For patients with metastatic
RCC, the most appropriate period for surgical treatment has been lost,
and systemic therapy is the only remedy [21]. Systemic therapy mainly
includes immunotherapy, targeted therapy, and chemotherapy. How-
ever, RCC is generally resistant to chemotherapeutic drugs. Currently,
only few of the potentially available chemotherapeutic drugs are used
in patients who have failed to respond to immunotherapy or have tu-
mors that are resistant to targeted therapy. In addition, patients with
more pronounced sarcomatoid changes are also recommended to use
chemotherapy [22]. The main modes of chemotherapy include single-
agent chemotherapy, combination chemotherapy, chemotherapy com-
bined with immunotherapy and chemotherapy combined with targeted
therapy [23]. However, the effects of chemotherapy are not truly sa-
tisfactory, and because of the different research designs, chemotherapy,
and targeted therapy cannot be compared head-to-head. The main
reason for the insensitivity of RCC to chemotherapy is the over-
expression of MDR-1, which increases the expression of P-gp and en-
hances its effect, so that the chemotherapy drug can no longer be
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Y. Zheng, et al.
Biomedicine&Pharmacotherapy116(2019)109001
accumulated intracellularly in the effective cytotoxic concentration [9].
We have also detected the relative high expression of P-gp in ACHN and
786-O cell lines by real-time quantitative polymerase chain reaction
(Supplementary Fig. 3). For a long time, scholars have been attempting
to increase the sensitivity of RCC to chemotherapy. Wang B. et al. [24]
used tigecycline to interfere with the translation of mitochondrial DNA
in tumor cells, thereby affecting their respiration complex and re-
spiratory function to increase the sensitivity of RCC to chemotherapy.
Chen D. et al. [25] increased the sensitivity of RCC cells to 5-fluor-
ouracil by using atovaquone to inhibit mitochondrial respiration. Pre-
viously, our research team has found that cyanine-based NIRF dye IR-
780 can be efficiently absorbed by RCC cells [3]. If these moieties are
coupled with chemotherapeutic drugs, it may increase the stable con-
centration of intracellular chemotherapeutic drugs in RCC cells.
IR-780 is a heptamethylcyanine compound which is a cyanine-based
NIRF dye. It is stably absorbed by tumor cells through the OATP su-
perfamily transporters and has tumor imaging properties. It is widely
used in research on tumor diagnosis and treatment [26–28]. The OATP
superfamily is related to the solute carrier (SLCO) gene family. There
are 11 subtypes known in humans, which can be classified into 6 ca-
tegories. OATP1 includes OATP1A2, OATP1B1, OATP1B3, and
OATP1C1, the OATP2 family has two subtypes, OATP2A1 and
OATP2B1, and the OATP4 family includes OATP4A1 and OATP4C1.
OATP3A1, OATP5A1, and OATP6A1 are the only members of OATP3,
OATP5, and OATP6, respectively. These transmembrane proteins play
an important role in the absorption, distribution and excretion of hor-
mones, drugs, and exogenous materials, and are closely related to the
prognosis and treatment of tumors [29]. The expression of OATP in
RCC has been reported previously [30], which provides a theoretical
basis for the treatment of RCC with IR-780 coupled with chemother-
apeutic drugs.
The anticancer drug cabazitaxel is a paclitaxel-derived compound
that exerts an anti-tumor role mainly by inhibiting mitosis. It can bind
to tubulin, promote the assembly of microtubule dimers into micro-
tubules, stabilize the structure of microtubules, and keep cells in the
mitotic G2 and M phases, thus inhibiting mitosis and proliferation of
cancer cells [31]. At the same time, IR-780 and cabazitaxel are easy to
synthesize and modify, and the synthesized new compounds are
structurally stable and recalcitrant to decomposition. In previous stu-
dies, our team has chemically coupled IR-780 with abiraterone and
successfully applied it to the diagnosis and treatment of prostate cancer
[32]. Therefore, we combined IR-780 with cabazitaxel to make use of
the property of IR-780 to be transported into RCC cells through the
OATP family transporters. We hoped that the newly synthesized Caba-
780 can be efficiently absorbed by RCC and exert the anti-tumor effect
of cabazitaxel at the same time. Indeed, the new synthetic drug Caba-
780 was efficiently taken up by RCC cells and RCC tissues in vitro and in
vivo, and could be used in conjunction with an imaging system, de-
monstrating its diagnostic application prospects. As predicted, OATP
blocking agents blocked the absorption of Caba-780 by RCC cells,
which indicates that Caba-780 is also transported into cells through the
OATP family of transporters. In other words, the NIRF dye moiety of the
synthetic drug Caba-780 plays the role of a “ferryman”. In vitro, the new
drug Caba-780 exerted a strong lethal effect on tumor cells, which was
manifested by the inhibition of clonal growth, migration, and invasion
of RCC cells, as well as the increase in apoptosis. These effects were
stronger than those of IR-780 and cabazitaxel. Moreover, Caba-780
prolonged the G2 phases of RCC cells, which corresponded to the anti-
tumor mechanism of cabazitaxel. This also indicates that the cabazi-
taxel moiety of Caba-780 exerts the primary cytotoxic effect [31].
In our in vitro tests, Caba-780 had a certain cytotoxic effect on
normal HEK293 cells, as these cells can absorb a small amount of Caba-
780. In the toxicity assay, the cells were incubated with the drugs for
24 h, and the accumulation of a small amount was also toxic. However,
it is undeniable that the cytotoxicity of Caba-780 is stronger than that of
IR-780 and cabazitaxel in two kinds of RCC cells. The concentration of
the drug added in the cell experiments was consistent, but the dis-
tribution of the drug in the animal experiments was inconsistent. In the
in vivo experiment, Caba-780 was able to accumulate in the tumor
tissue, and the acute toxicity assay confirmed that Caba-780 had no
effect on the main organs. These results confirmed that the targeted
imaging and tumor-cytotoxic effect of Caba-780 can be sustained, and
also confirmed that this new synthetic drug has certain application
prospects in the diagnosis and treatment of RCC. The IC50 of cabazi-
taxel was not obtained because the RCC was too insensitive to che-
motherapy drugs, and cabazitaxel could not kill RCC cells within a
limited concentration range. Mizutani K. et al. [33] have reported that
cabazitaxel was effective in RCC cell lines, but they only inoculated 500
cells per well in the toxicity assay. At such low cell density, the cells are
very sensitive, so their reported IC50 value was very low. We think that
this is not an appropriate approach and we believe that the efficacy of
cabazitaxel alone in the treatment of RCC is not ideal, after all, there are
no reports on clinically relevant application. Although cabazitaxel does
not exert toxicity, it has an effect on migration and clonogenicity of
RCC cells. This interesting phenomenon is due to the ability of caba-
zitaxel to inhibit the process of epithelial-mesenchymal transition of
tumor cells, that allowing tumor cells to metastasize, which is shown by
their migration and clonogenicity [34].
Further research is needed to confirm the findings of this study.
Firstly, Caba-780 was effective in RCC as shown in our in vitro and in
vivo experiments, but the mechanism of action of Caba-780 remains
unknown. One hypothesis is that in the lysosomal environment where
the pH is approximately 5, the ester bonds in caba-780 break and ca-
bazitaxel can be released to function. Moreover, esterase is highly ex-
pressed in cancer cells, which can also break ester bonds [35]. Sec-
ondly, how Caba-780 is metabolized in the body, through the
hepatointestinal or the urinary system remains unknown. Thirdly, the in
vitro fluorescence intensity of Caba-780 is weaker than that of IR-780,
which results in the weaker intensity of Caba-780 than that of IR-780 in
vivo imaging. However, it does not affect the imaging and cytotoxicity
of Caba-780 for the diagnosis and treatment of RCC. Finally, the OATP
family is expressed in prostate cancer, bladder cancer, and many other
tumors [36,37]. Therefore, we will further investigate the biological
effects of Caba-780 on other tumors in future studies.
5. Conclusion
In summary, the new synthetic drug Caba-780 was efficiently ab-
sorbed by RCC cells. The cytotoxic effect appears to be mediated by
inhibiting the clonal growth, migration and invasion of RCC cells,
promoting their apoptosis, and prolonging the G2 and S cell cycle
stages. In the tumor-bearing nude mouse model, Caba-780 was effi-
ciently absorbed by the tumor, which makes it useful for imaging in
addition to its cytotoxic effects. Thus, Caba-780 has potential applica-
tion prospects in both the diagnosis and treatment of RCC. Taken to-
gether, the results indicate that coupling NIRF dyes with chemother-
apeutic drugs may make it possible to treat RCC in the near future.
Conflicts of interest
The authors have declared no conflicts of interest.
Funding
This work was supported by the Military medical innovation project
[grant number 16CXZ023], the Key Science and Technology Program of
Shaanxi Province [grant number 2014k11‑03-05-01] and the Xijing
Hospital subject booster plan translational medicine research projects
[grant number XJZT13Z05].
11

Y. Zheng, et al.
Biomedicine&Pharmacotherapy116(2019)109001
Acknowledgement
genes in renal cell carcinomas and normal kidney, J. Urol. 156 (1996) 506–511.
[16] H. Sato, H. Senba, N. Virgona, K. Fukumoto, T. Ishida, H. Hagiwara, E. Negishi,
K. Ueno, H. Yamasaki, T. Yano, Connexin 32 potentiates vinblastine-induced cy-
totoxicity in renal cell carcinoma cells, Mol. Carcinog. 46 (2007) 215–224.
[17] C.C. Liang, A.Y. Park, J.L. Guan, In vitro scratch assay: a convenient and in-
expensive method for analysis of cell migration in vitro, Nat. Protoc. 2 (2007)
329–333.
The authors thank laboratory staff Dr.Tianshu Du, Yan Zhao, Furong
Wang and Lirong Ma for their hard work in maintaining the experi-
mental environment and animal feeding for a long time. Dr. Zheng also
would like to thank his wife Sally Su for her love support and language
modification.
[18] C.R. Justus, N. Leffler, M. Ruiz-Echevarria, L.V. Yang, In vitro cell migration and
invasion assays, J. Vis. Exp. (2014).
[19] M. Gao, B.Z. Tang, Fluorescent sensors based on aggregation-induced emission:
recent advances and perspectives, ACS Sens. 2 (2017) 1382–1399.
[20] U. Capitanio, F. Montorsi, Renal cancer, Lancet 387 (2016) 894–906.
[21] T.K. Choueiri, R.J. Motzer, Systemic therapy for metastatic renal-cell carcinoma, N.
Engl. J. Med. 376 (2017) 354–366.
[22] E. Diamond, A.M. Molina, M. Carbonaro, N.H. Akhtar, P. Giannakakou,
S.T. Tagawa, D.M. Nanus, Cytotoxic chemotherapy in the treatment of advanced
renal cell carcinoma in the era of targeted therapy, Crit. Rev. Oncol. Hematol. 96
(2015) 518–526.
[23] S. Buti, M. Bersanelli, A. Sikokis, F. Maines, F. Facchinetti, E. Bria, A. Ardizzoni,
G. Tortora, F. Massari, Chemotherapy in metastatic renal cell carcinoma today? A
systematic review, Anticancer Drugs 24 (2013) 535–554.
[24] B. Wang, J. Ao, D. Yu, T. Rao, Y. Ruan, X. Yao, Inhibition of mitochondrial trans-
lation effectively sensitizes renal cell carcinoma to chemotherapy, Biochem.
Biophys. Res. Commun. 490 (2017) 767–773.
[25] D. Chen, X. Sun, X. Zhang, J. Cao, Targeting mitochondria by anthelmintic drug
atovaquone sensitizes renal cell carcinoma to chemotherapy and immunotherapy, J.
Biochem. Mol. Toxicol. 32 (2018) e22195.
[26] C. Yue, P. Liu, M. Zheng, P. Zhao, Y. Wang, Y. Ma, L. Cai, IR-780 dye loaded tumor
targeting theranostic nanoparticles for NIR imaging and photothermal therapy,
Biomaterials 34 (2013) 6853–6861.
[27] Y. Li, Q. Zhou, Z. Deng, M. Pan, X. Liu, J. Wu, F. Yan, H. Zheng, IR-780 dye as a
sonosensitizer for sonodynamic therapy of breast tumor, Sci. Rep. 6 (2016) 25968.
[28] S.K. Rajendrakumar, N.C. Chang, A. Mohapatra, S. Uthaman, B.I. Lee, W.B. Tsai,
I.K. Park, A lipophilic IR-780 dye-encapsulated zwitterionic polymer-lipid micellar
nanoparticle for enhanced photothermal therapy and NIR-Based fluorescence
imaging in a cervical tumor mouse model, Int. J. Mol. Sci. 19 (2018).
[29] A. Obaidat, M. Roth, B. Hagenbuch, The expression and function of organic anion
transporting polypeptides in normal tissues and in cancer, Annu. Rev. Pharmacol.
Toxicol. 52 (2012) 135–151.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.biopha.2019.109001.
References
[1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2019, CA Cancer J. Clin. 69
(2019) 7–34.
[2] I.S. Gill, M. Aron, D.A. Gervais, M.A. Jewett, Clinical practice. Small renal mass, N.
Engl. J. Med. 362 (2010) 624–634.
[3] X. Yang, C. Shao, R. Wang, C.Y. Chu, P. Hu, V. Master, A.O. Osunkoya, H.L. Kim,
H.E. Zhau, L. Chung, Optical imaging of kidney cancer with novel near infrared
heptamethine carbocyanine fluorescent dyes, J. Urol. 189 (2013) 702–710.
[4] G. Bergers, D. Hanahan, Modes of resistance to anti-angiogenic therapy, Nat. Rev.
Cancer 8 (2008) 592–603.
[5] W. Lilleby, S.D. Fossa, Chemotherapy in metastatic renal cell cancer, World J. Urol.
23 (2005) 175–179.
[6] R. Petrioli, L. Paolelli, E. Francini, S. Marsili, A. Pascucci, A. Sciandivasci, G. de
Rubertis, G. Barbanti, A. Manganelli, F. Salvestrini, G. Francini, Capecitabine as
third-line treatment in patients with metastatic renal cell carcinoma after failing
immunotherapy, Anticancer Drugs 18 (2007) 817–820.
[7] I. Tsimafeyeu, L. Demidov, G. Kharkevich, N. Petenko, V. Galchenko, I. Sinelnikov,
U. Naidzionak, Phase II, multicenter, uncontrolled trial of single-agent capecitabine
in patients with non-clear cell metastatic renal cell carcinoma, Am. J. Clin. Oncol.
35 (2012) 251–254.
[8] P.J. Van Veldhuizen, M. Hussey, P.J. Lara, P.C. Mack, R. Gandour-Edwards,
J.I. Clark, M.K. Lange, D.E. Crawford, A phase ii study of gemcitabine and cape-
citabine in patients with advanced renal cell cancer: southwest Oncology Group
Study S0312, Am. J. Clin. Oncol. 32 (2009) 453–459.
[9] N. Walsh, A. Larkin, S. Kennedy, L. Connolly, J. Ballot, W. Ooi, G. Gullo, J. Crown,
M. Clynes, L. O’Driscoll, Expression of multidrug resistance markers ABCB1 (MDR-
1/P-gp) and ABCC1 (MRP-1) in renal cell carcinoma, BMC Urol. 9 (2009) 6.
[10] R. Vasko, G.A. Mueller, A.K. von Jaschke, A.R. Asif, H. Dihazi, Impact of cisplatin
administration on protein expression levels in renal cell carcinoma: a proteomic
analysis, Eur. J. Pharmacol. 670 (2011) 50–57.
[11] M.M. Baldewijns, I.J. van Vlodrop, P.B. Vermeulen, P.M. Soetekouw, M. van
Engeland, A.P. de Bruine, VHL and HIF signalling in renal cell carcinogenesis, J.
Pathol. 221 (2010) 125–138.
[30] T. Nakanishi, I. Tamai, Putative roles of organic anion transporting polypeptides
(OATPs) in cell survival and progression of human cancers, Biopharm. Drug Dispos.
35 (2014) 463–484.
[31] G. Attard, A. Greystoke, S. Kaye, J. De Bono, Update on tubulin-binding agents,
Pathol Biol (Paris) 54 (2006) 72–84.
[32] X. Yi, J. Zhang, F. Yan, Z. Lu, J. Huang, C. Pan, J. Yuan, W. Zheng, K. Zhang, D. Wei,
W. He, J. Yuan, Synthesis of IR-780 dye-conjugated abiraterone for prostate cancer
imaging and therapy, Int. J. Oncol. 49 (2016) 1911–1920.
[33] K. Mizutani, M. Tomoda, Y. Ohno, H. Hayashi, Y. Fujita, K. Kawakami,
K. Kameyama, T. Kato, T. Sugiyama, Y. Itoh, M. Ito, T. Deguchi, Effects of
Cabazitaxel in renal cell carcinoma cell lines, Anticancer Res. 35 (2015)
6671–6677.
[34] S.K. Martin, H. Pu, J.C. Penticuff, Z. Cao, C. Horbinski, N. Kyprianou,
Multinucleation and Mesenchymal-to-Epithelial Transition Alleviate Resistance to
Combined Cabazitaxel and Antiandrogen Therapy in Advanced Prostate Cancer,
Cancer Res. 76 (2016) 912–926.
[35] H. Dong, L. Pang, H. Cong, Y. Shen, B. Yu, Application and design of esterase-
responsive nanoparticles for cancer therapy, Drug Deliv. 26 (2019) 416–432.
[36] N. Thakkar, A.C. Lockhart, W. Lee, Role of organic anion-transporting polypeptides
(OATPs) in Cancer therapy, AAPS J. 17 (2015) 535–545.
[12] A.M. Roberts, I.R. Watson, A.J. Evans, D.A. Foster, M.S. Irwin, M. Ohh, Suppression
of hypoxia-inducible factor 2alpha restores p53 activity via Hdm2 and reverses
chemoresistance of renal carcinoma cells, Cancer Res. 69 (2009) 9056–9064.
[13] H. Zhu, Z. Wang, Q. Xu, Y. Zhang, Y. Zhai, J. Bai, M. Liu, Z. Hui, N. Xu, Inhibition of
STAT1 sensitizes renal cell carcinoma cells to radiotherapy and chemotherapy,
Cancer Biol. Ther. 13 (2012) 401–407.
[14] I. Kausch, H. Jiang, B. Thode, C. Doehn, S. Kruger, D. Jocham, Inhibition of bcl-2
enhances the efficacy of chemotherapy in renal cell carcinoma, Eur. Urol. 47 (2005)
703–709.
[37] A. Obaidat, M. Roth, B. Hagenbuch, The expression and function of organic anion
transporting polypeptides in normal tissues and in cancer, Annu. Rev. Pharmacol.
Toxicol. 52 (2012) 135–151.
[15] W.J. Kim, Y. Kakehi, H. Kinoshita, S. Arao, M. Fukumoto, O. Yoshida, Expression
patterns of multidrug-resistance (MDR1), multidrug resistance-associated protein
(MRP),glutathione-S-transferase-pi (GST-pi) and DNA topoisomerase II (Topo II)
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