Mitochondria-targeted prostate cancer therapy using a near-infrared fluorescence dye–monoamine oxidase A inhibitor conjugate

Article abstract of DOI:10.1016/j.jconrel.2018.04.038

Prostate cancer (PCa) is the most frequent malignant cancer among men in the USA, leading to substantial morbidity and mortality, while the existing treatments have restricted therapeutic benefits for patients with hormone-refractory PCa (HRPC) and metast

Full text of DOI:10.1016/j.jconrel.2018.04.038

Accepted Manuscript
Mitochondria-targeted prostate cancer therapy using a near-
infrared fluorescence dye–monoamine oxidase A inhibitor
conjugate
Qingzhi Lv, Xiaoguang Yang, Menglin Wang, Jincheng Yang,
Zhilin Qin, Qiming Kan, Haotian Zhang, Yongjun Wang, Dun
Wang, Zhonggui He
PII:
DOI:
Reference:
S0168-3659(18)30222-0
doi:10.1016/j.jconrel.2018.04.038
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Journal of Controlled Release
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Accepted date:
15 January 2018
23 March 2018
18 April 2018
Please cite this article as: Qingzhi Lv, Xiaoguang Yang, Menglin Wang, Jincheng Yang,
Zhilin Qin, Qiming Kan, Haotian Zhang, Yongjun Wang, Dun Wang, Zhonggui He ,
Mitochondria-targeted prostate cancer therapy using a near-infrared fluorescence
dye–monoamine oxidase A inhibitor conjugate. The address for the corresponding author
was captured as affiliation for all authors. Please check if appropriate. Corel(2018),
doi:10.1016/j.jconrel.2018.04.038
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ACCEPTED MANUSCRIPT
Mitochondria-Targeted Prostate Cancer Therapy Using a Near-Infrared Fluorescence
Dye−Monoamine Oxidase A Inhibitor Conjugate
Qingzhi Lva,b, Xiaoguang Yangc, Menglin Wanga, Jincheng Yangc, Zhilin Qina, Qiming Kand,
Haotian Zhangd, Yongjun Wang,a,* Dun Wang,c,* Zhonggui Hea,*
aWuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, China
bSchool of Pharmacy, Binzhou Medical University, Yantai 264003, China
cKey Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, China
dSchool of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University,
Shenyang 110016, China
*Corresponding authors.
Abstract
Prostate cancer (PCa) is the most frequent malignant cancer among men in the USA,
leading to substantial morbidity and mortality, while the existing treatments have restricted
therapeutic benefits for patients with hormone-refractory PCa (HRPC) and metastatic PCa.
Recent studies show that advanced PCa exhibits an increase in the expression of monoamine
oxidase A (MAOA) which is a mitochondria enzyme, and MAOA activity inhibition could
restrict metastasis and extend mice survival in PCa xenografts. These findings suggest MAOA
can be a potential target to treat PCa. For this reason, we identify and synthesize a near-
infrared fluorescence (NIRF) heptamethine dye−MAOA inhibitor conjugate (NIR-INH) for
simultaneous PCa imaging, targeting and therapy. The conjugate combines a NIRF dye for
mitochondria targeting with the MAOA inhibitor isoniazid (INH). NIR-INH exhibits specific
targeting in PCa xenografts and markedly inhibited tumor growth. Furthermore, there is no
obvious toxicity with NIR-INH treatment, which is a remarkable superiority towards
traditional chemotherapy. These results indicate that NIR-INH has PCa targeting, imaging and
high anticancer effectiveness, suggesting it is a potentially valuable image-guided anti-tumor
strategy.
Keywords: Isoniazid; Mitochondria; Monoamine Oxidase A; Near-Infrared Fluorescence
Dyes; Prostate Cancer
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1. Introduction
According to a report from the American Cancer Society in 2016, PCa is the most
frequent malignant cancer among American men and the second-leading cause of cancer
mortality [1-3]. However, several therapies such as hormonal therapy, radiation therapy and
radical prostatectomy are effective in curing localized PCa, they have restricted benefits for
patients with hormone-refractory PCa (HRPC) and metastatic PCa. In addition, HRPC have
proven to be resistant to chemotherapy [4-6]. Therefore, there is a critical need for new and
effective mechanism-based PCa therapy with minimal adverse effects.
Monoamine oxidase A (MAOA) is a mitochondrial enzyme that degrades
neurotransmitters such as serotonin and dopamine by oxidative deamination [7, 8]. The
process generates hydrogen peroxide as a by-product, a main source of ROS, which can cause
tumor initiation and progression [9-12]. Previous studies show that MAOA could cause
epithelial-to-mesenchymal transition (EMT) through the production of ROS that stabilize
HIF1α [9, 13]. Additionally, MAOA expression increases after exposure to cytotoxic drug
and leads to chemotherapy resistance [14]. Wu et al. find that increased MAOA expression is
associated with PCa progression and metastasis. MAOA inhibition dramatically restricts
metastasis and extended survival in PCa xenograft mouse models [15]. These findings suggest
MAOA can be a potential target to treat metastatic and advanced PCa.
Given these results, we synthesized a novel compound that combines a NIRF dye and an
MAOA inhibitor to selectively accumulate in PCa cells. The NIRF dye MHI-148 was
conjugated with the terminal amino group of isoniazid (INH). INH, originally synthesized in
1951, is a small molecule inhibitor of MAOA and remains the most powerful first-line anti-
tuberculosis medicines. NIRF heptamethine cyanine dyes are utilized as a useful tool for
personalized oncology to simultaneously target cancer and noninvasively image tumors
without chemical conjugation. They retain in a variety of cancer cells and accumulate in
cellular mitochondria [16-19]. Tumor cells usually possess higher negative mitochondrial
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transmembrane potentials than normal cells and some lipophilic cations preferentially
accumulate in cellular mitochondria of tumor cells. The higher mitochondrial transmembrane
potential in tumor cells may play a vital role in the preferential accumulation of these NIRF
dyes because these dyes are lipophilic cationic molecules [16]. Further studies demonstrated
that the preferential tumor accumulation of NIRF dyes were mediated by tumor hypoxia and
activated HIF1α/organic anion-transporting polypeptides (OATPs) signaling axis [20].
OATPs are overexpressed on the surface of many tumor cells, including human PCa.
OATP1B3, a representative OATP member, may play a key role in controlling the
accumulation of NIRF dyes (IR-780, IR-783, and MHI-148) in tumor cells [20-23]. In our
study, we use numbers of in vitro and in vivo PCa models to evaluate the tumor imaging,
targeting and therapy potential of MHI-148 dye and NIR-INH conjugate for PCa.
2. Materials and Methods
2.1. Materials
Isoniazid (INH) and 3-methyl-2-butanone were purchased from Aladdin (Shanghai,
China). Phenylhydrazine and 6-bromohexanoic acid were purchased from Meryer (Shanghai,
China). F12K media and FBS were purchased from Gibco (MD, USA). PC-3 cells were
obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). DAPI,
Hoechst 33258, and MitoTracker Green were obtained from Beyotime (Shanghai, China).
Thiazolyl blue tetrazoliumbromide (MTT) and trypsin-EDTA were obtained from Sigma-
Aldrich (MO, USA). All other chemicals and reagents applied were analytical or HPLC grade.
2.2. Synthesis and structural characterization of MHI-148-isoniazid (NIR-INH)
2,3,3-trimethyl-3H-indole (1). Phenylhydrazine (10 g, 69.4 mmol) and 3-methyl-2-
butanone (17.9 g, 208.3 mmol) were added to acetic acid, and the mixture was stirred for 4
hours at 120 °C, cooled to room temperature (rt) and filtered to remove by-products. The
solvent was removed in vacuo. The residue was purified by a silica gel to give 1 (9.4 g, 85%).
MS (ESI) m/z: 182[M+Na]+.
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1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium bromide (2). A solution of 1 (2 g,
12.6 mmol) and 6-bromohexanoic acid (9.76 g, 50.3 mmol) in acetonitrile was refluxed for 40
hours under nitrogen. The solvent was removed in vacuo. The residue was suspended in ethyl
acetate and then filtered to give 2 (3.27 g, 73.5%). MS (ESI) m/z: 274[M-Br]+.
(E)-2-chloro-3-(hydroxymethylene)cyclohex-1-enecarbaldehyde (3). Phosphorous
oxychloride (32 mL, 5 M in DCM, 160 mmol) was added to a solution of DMF/DCM mixture
(1:1) in an ice bath. Then, cyclohexanone (5 g, 50 mmol) was added, and the resulting
mixture was vigorously stirred and heated to reflux for 3 hours, cooled to rt and then poured
into ice-cooled water. The mixture was kept at 2-6°C overnight and then filtered to obtain
compound 3 (4.7 g, 55%). 1H NMR (400MHz, DMSO-d6 ) δ/ppm:1.58 (2H, m),2.63 (4H,
s),7.52 (1H, s),10.14 (1H, d),10.86 (1H, s).
1-(5-carboxypentyl)-2-(-2-(3-(-2-(1-(5-carboxypentyl)-3,3-dimethylindolin-2-
ylidene)ethylidene)-2-chlorocyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-3H-indol-1-ium
bromide (MHI-148). Compound 2 (500 mg, 1.42 mmol) was added to a solution of
compound 3 (122 mg, 0.71 mmol) and sodium acetate (58 mg, 0.71 mmol) in acetic anhydride.
The reaction mixture was heated at 70°C under vigorous stirring for 40 min, and then cooled
to rt. The mixture was added to water and stirred for 3 hours, followed by extraction with
ethyl acetate. The organic layer was dried over Na2SO4 and concentrated. The residue was
purified by chromatography on silica gel to yield MHI-148 (380 mg, 70%). MS (ESI)
m/z:683 [M-Br]+. 1H NMR (600 MHz, CDCl3) δ 9.80-9.36 (br, 2H), δ 8.34 (d, J = 13.9 Hz,
2H), 7.39 (d, J = 7.9 Hz, 4H), 7.24 (t, J = 7.5 Hz, 2H), 7.20 (d, J = 7.9 Hz, 2H), 6.18 (d, J =
14.0 Hz, 2H), 4.14 (t, J = 7.1 Hz, 4H), 2.74 – 2.68 (m, 4H), 2.48 (t, J = 7.2 Hz, 4H), 2.02 –
1.96 (m, 2H), 1.86 (dt, J = 14.8, 7.5 Hz, 4H), 1.76 (dt, J = 14.8, 7.4 Hz, 4H), 1.71 (s, 12H),
1.59 – 1.51 (m, 4H).
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2-[2-[3-[2-[1-(5-carboxypentyl)-1,3-dihydro-3,3-dimethyl-2H-indol-2-
ylidene]ethylidene]-2-chloro-1-cyclohexen-1-yl]ethenyl]-1-(6-(2-isonicotinoylhydrazinyl)-
6-oxohexyl)-3,3-dimethyl-3H-indolium bromide (NIR-INH). PyBOP (263 mg, 0.506
mmol) was added to a solution of MHI-148 (379 mg, 0.506 mmol) in DCM in an ice-salt bath.
The mixture was allowed to come to rt. Isoniazid (58 mg, 0.506 mmol) was added, and the
mixture was stirred for additional 15 hours. The mixture was filtered, evaporated under
reduced pressure and purified by preparative TLC to yield NIR-INH (153 mg, 34.3%). MS
(ESI) m/z:802.5 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 8.63 – 8.57 (br, 2H), 8.27 (d, J =
14.0 Hz, 1H), 8.17 (d, J = 13.6 Hz, 1H), 7.87 – 7.80 (br, 2H), 7.77 – 7.70 (br, 1H), 7.64 – 7.57
(br, 1H), 7.38 – 7.34 (m, 1H), 7.31 (d, J = 7.2 Hz, 2H), 7.18 (d, J = 7.4 Hz, 1H), 7.14 (d, J =
8.6 Hz, 2H), 7.06 – 7.01 (m, 2H), 6.27 (d, J = 13.7 Hz, 1H), 5.97 (d, J = 13.0 Hz, 1H), 4.12 –
4.06 (br, 2H), 3.95 – 3.89 (br, 2H), 2.55 – 2.50 (br, 2H), 2.47 – 2.41 (br, 2H), 2.40 – 2.33 (br,
2H), 2.32 – 2.25 (m, 2H), 1.89 – 1.79 (br, 2H), 1.78 – 1.71 (br, 4H), 1.67 (d, J = 9.6 Hz, 12H),
1.63 (s, 2H), 1.60 (d, J = 4.4 Hz, 2H), 1.56 (d, J = 5.6 Hz, 2H), 1.44 – 1.40 (m, 2H).
2.3. Determination of optical properties
MHI-148 and NIR-INH were dissolved in methanol (MeOH) and 100% FBS at a
concentration of 10 μM. The fluorescence and UV-vis absorbance spectra were determined,
respectively. For the serum stability test, MHI-148 and NIR-INH were incubated in 100%
FBS at 37 °C for 12 h. The absorbance and fluorescence intensities were determined using a
microplate reader (Thermo Scientific, USA). The photoluminescence was determined using
an IVIS imaging system (PerkinElmer, USA).
2.4. Cell culture
The PC-3 human PCa cells were cultured in F12K media with 10% FBS, 100U mL-1
penicillin, and 100 μg mL-1 streptomycin. Cells were grown in a humidified 5% CO2
atmosphere at 37 °C.
2.5. Cytotoxicity evaluation
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The cytotoxicity of INH, MHI-148, and NIR-INH was evaluated by MTT assay. Briefly,
PC-3 cells were incubated in 96-well plates at a density of 3000 cells/well for 12 h. Then the
cells were exposed to serial dilutions of INH, MHI-148, or NIR-INH for 48 h. After
incubation, 20 μL of MTT (5 mg mL-1) solution was added and the plates were incubated for
4 h at 37 °C. Then, the medium was replaced with 200 μL of DMSO to dissolve the formed
formazan crystals. The absorbance was determined at 570 nm with a microplate reader
(Thermo Scientific, USA).
2.6. Apoptosis assay
AV/PI detection kit (KeyGen Biotech, China) and flow cytometer (Becton Dickinson,
USA) were utilized to evaluate cell apoptosis. Briefly, PC-3 cells in six-well plates were
incubated with 20 μM INH, MHI-148 or NIR-INH at 37 °C for 24 h. Thereafter, cells were
stained with AV and PI for 15 min in the dark. In the end, cells were diluted with binding
buffer and analyzed by flow cytometry.
2.7. Subcellular localization and uptake assay
PC-3 cells were seeded on cover glass at a density of 2 ×105 cells/well for 24 h. Then
cells were incubated with 20 μM MHI-148 or NIR-INH at 37 °C for 1 h and washed thrice
with PBS. The cells were treated with MitoTracker Green for 30 min to specifically stain
mitochondria. The nuclei were stained with Hoechst 33258. Finally, the cells were observed
by confocal laser scanning microscopy (CLSM, Nikon Corp., Japan).
Cellular uptake of MHI-148 and NIR-INH was further quantified by flow cytometry.
Cells were incubated with 20 μM MHI-148 or NIR-INH at 37 °C or 0 °C for 1 h. The cells
were washed, harvested, and resuspended in PBS. Fluorescence intensity was analyzed on a
flow cytometer with 633 nm excitation and 650 nm emission for MHI-148 and NIR-INH. To
determine cellular uptake mechanism, PC-3 cells were pretreated with 250 μM BSP (an
OATP competitive inhibitor) for 1 h, and then incubated with 20 μM MHI-148 or NIR-INH
for 1 h at 37 °C.
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2.8. Animal and tumor xenograft model
All animal studies were carried out in accordance with the Guide for Care and Use of
Laboratory Animal of Shenyang Pharmaceutical University. The tumor xenograft models
were established by subcutaneously injecting 5×106 PC-3 cells in 50% Matrigel (BD
Biosciences, USA) into the armpit region of athymic nude BALB/C male mice.
2.9. In vivo biodistribution
Athymic nude mice bearing PC-3 tumor xenografts were used to investigate in vivo
biodistribution of INH, MHI-148, and NIR-INH. Mice with subcutaneous tumors of 100-150
mm3 were administered through tail vein injection of INH, MHI-148, or NIR-INH at 2.5 μM
kg-1. The mice were sacrificed at 48 h, and ex vivo imaging signals of major organs and
tumors were detected using an IVIS imaging system (PerkinElmer, USA).
2.10. In vivo tumor inhibitory effects and toxicity
To determine the therapeutic effect of NIR-INH, we used mouse tumor xenografts
bearing PC-3 tumors. When the tumor size reached 75-100 mm3, the nude mice were
randomied to 4 groups (n=5): (1) saline (the control group), (2) INH, (3) MHI-148, (4) NIR-
INH. These preparations were injected through tail veins (37.5 μM kg-1) at a 3-day interval for
15 days. Tumor volume and body weight were measured. Mice were sacrificed 48 h post-final
injection, and blood was collected. The serum levels of blood urea nitrogen (BUN), creatinine
(CREA), aspartate transaminase (AST), and alanine transaminase (ALT) were determined to
assess hepatic and renal function. The serum prostate-specific antigen (PSA) levels were
measured by an ELISA kit (Cusabio Biotech, China) according to the manufacturer’s
protocols.
The tumor and major organs were harvested and sectioned for further pathological
assessment. The dissected tumors were accurately weighed to calculate the tumor burden, and
the tumor inhibition rate (TIR) was calculated as followed: TIR%=(1-Wsample/Wcontrol)×100.
Tumor cell apoptosis was assessed by TUNEL assay, using an apoptosis detection kit
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(KeyGen Biotech, China) in accordance with the manufacturer’s instructions. The nuclei of
tumor sections were stained by DAPI. All stained samples were visualized with a confocal
microscope (Nikon Corp., Tokyo, Japan). The formalin-embedded tissues were stained with
H&E to observe the systematic toxicity of major organs.
2.11. Statistical analysis
Data are expressed as mean ± standard deviation. A one-way ANOVA was used to
determine the significance in the experiments. Statistical differences were significant at P <
0.05 and highly significant at P < 0.01.
3. Results and Discussions
3.1. Chemical synthesis and structure characterization of MHI-148 and NIR-INH
MHI-148 and NIR-INH were synthesized in a sequence of procedures (Figure 1). The
preparation of the compound 2,3,3-trimethyl-3H-indole (1) was obtained from
phenylhydrazine hydrochloride by a 3-methyl-2-butanone mediated ring-close condensation,
which was subsequently alkylated with 6-bromohexanoic acid to obtain 1-(5-carboxypentyl)-
2,3,3-trimethyl-3H-indol-1-ium bromide (2). Cyclohexanone underwent a Vilsmeier-Haack
protocol to afford (E)-2-chloro-3-(hydroxymethylene)cyclohex-1-ene-1-carbaldehyde (3),
which was then condensed with intermediate 2 to obtain MHI-148 with a total yield 24.1% in
4 steps. The structure of MHI-148 was confirmed by 1H NMR and MS (Figure S1, S2). The
target molecule, NIR-INH, was synthesized by a condensation of MHI-148 and INH in one
step. The compound was purfied by preparative TLC, and its chemical structure was
characterized by 1H NMR and MS (Figure S3, S4). The chemical structures of MHI-148 and
NIR-INH are illustrated in Figure S5.
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Figure 1. Synthesis of MHI-148 and NIRF Dye−MAOA inhibitor Conjugate NIR-INH.
3.2. Optical properties of MHI-148 and NIR-INH
The optical properties of MHI-148 and NIR-INH were investigated in methanol and
100% FBS, respectively (Figure 2 A, B, D). The absorption peak of NIR-INH was at 782 nm
in methanol and 792 nm in FBS. While the emission peak was at 802 nm in methanol and 817
nm in FBS, providing 20-30 nm Stokes shifts of NIR-INH. MHI-148 displayed similar optical
properties. Previous studies have demonstrated that the introduction of a cyclohexenyl ring
substitution in the middle of polymethine chain can dramatically increase photochemical
stability and fluorescence efficiency of carbocyanine dyes [16, 24]. Furthermore, the
fluorescence intensity is markedly increased upon the binding of carbocyanine dyes with
nucleic acids or proteins because of the rigidization of the cyanine dyes [24]. In the current
study, a photostability study of NIRF dyes in FBS showed that the fluorescence intensity of
MHI-148 and NIR-INH maintained at a stable level for 12 h. This indicates that MHI-148 and
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NIR-INH are fairly stable in serum (Figure 2 C), which suggests they have a promising
potential for use in biomedical imaging.
Figure 2. Optical properties of MHI-148 and NIR-INH. (A) The absorption spectra of MHI-
148 and NIR-INH in methanol and FBS, respectively. (B) The emission spectra of MHI-148
and NIR-INH in methanol and FBS, respectively. (C) Fluorescence stability of 10 μM MHI-
148 and NIR-INH in FBS at 37 °C for 12 h. (D) Color and fluorescence image of MHI-148
and NIR-INH in FBS (F, left) and methanol (M, right) at a concentration of 10 μM.
3.3. In vitro anticancer activity and apoptosis
The anticancer activity of INH, NIR-INH, and MHI-148 against PC-3 cells was
investigated by an MTT assay. As shown in Figure 3 A, INH incubation indicated no obvious
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cytotoxicity at a concentration of 200 μM (cell viability=87.21%). NIR-INH displayed higher
efficacy when compared to MHI-148 at the concentration range of 0.5-100 μM (Figure 3 A).
Evaluation of IC50 values indicated that the conjugate NIR-INH (IC50=33.806 μM) had 4.2-
fold higher cytotoxicity than the MHI-148 dye (IC50=141.461 μM) in inhibiting PC-3 cells
growth. NIR-INH exhibited concentration-dependent cytotoxicity on PC-3 cells and nearly
eliminated all cells at a concentration of 100 μM.
Fluorescence-activated cell sorting (FACS) analysis was utilized to measure cell
apoptosis (Figure 3 B). NIR-INH (40.90%) treatment caused a robust increase in apoptotic
cells, as compared to MHI-148 (14.55%) and INH (10.55%) treatment. The modest
cytotoxicity and cell apoptosis of MHI-148 may due to its accumulation in the mitochondria
of PC-3 cells, and disruption of mitochondrial activities [19]. Wang et al. demonstrated that
NIRF dyes (IR 780 and MHI-148) could retain in the mitochondria of cancer cells, increase
ROS production and decrease mitochondrial membrane potential, induce cancer cells
apoptosis [19, 22]. Some studies demonstrated that INH-type hydrazone and N’-acylated
derivatives are more efficient and less hepatotoxic than INH, due to blockage of the terminal
amino group [25-27]. Therefore, NIR-INH may possess higher MAOA inhibition efficacy
than INH with the terminal hydrazide group conjugated with MHI-148. The preferential cell
uptake mediated by OATPs and increased efficacy derived of INH N’-acylation result in the
increased anticancer efficacy of NIR-INH. MAOA inhibitor INH showed negligible
cytotoxicity, which probably attribute to its poor passive diffusion into PC-3 cells despite its
MAOA inhibitory activity. (log P = -0.69).
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Figure 3. Cell viability and apoptosis studies of MHI-148 and NIR-INH. (A) In vitro
antitumor activity of MHI-148 and NIR-INH against PC-3 cells for 48 h (mean±SD, n=3). (B)
Apoptosis rates of INH, MHI-148, and NIR-INH.
3.4. Subcellular localization of MHI-148 and NIR-INH
Recent reports suggested that MHI-148 could selectively accumulate in various types of
cancer cells. To investigate subcellular localization, PC-3 cells were co-stained with
MitoTracker and MHI-148/NIR-INH conjugates. The CLSM images indicated that MHI-148
and NIR-INH accumulated in PC-3 cells and localized in cellular mitochondrias, as measured
by colocalized with MitoTracker (Figure 4 A). Mitochondria are vital energy-producing
cellular organelles and play a key role in cell apoptosis. Therefore, mitochondria is a preferred
subcellular target suitable for cancer treatment. Tumor cells usually possess higher
mitochondrial membrane potentials than normal cells and some lipophilic cations
preferentially accumulate in cellular mitochondria of tumor cells because of the higher
negative membrane potentials of mitochondria. The higher mitochondrial transmembrane
potential in tumor cells may play a vital role in the preferential accumulation of these NIRF
dyes [16].
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Wu et al. demonstrated the preferential tumor cellular uptake of NIRF dyes was
mediated by tumor hypoxia and activated HIF1α/OATPs signaling axis. OATP is
overexpressed on the surface of many tumor cells, including human PCa [20]. To examine
cellular uptake mechanism, PC-3 cells were incubated with NIR-INH/MHI-148 at 4 °C or
37 °C. The CLSM images revealed the uptake of NIR-INH and MHI-148 were markedly
decreased when incubated at 4 °C (Figure 4 B), suggesting the cellular uptake of MHI-148
and NIR-INH is an energy-dependent process. However, when PC-3 cells were pretreated
with BSP (a competitive inhibitor of OATPs), there was a significant decrease in the uptake
of NIR dyes, even though it was incubated at 37 °C (Figure 4 B). This result demonstrates
that the cell uptake of NIR-INH and MHI-148 is mainly mediated by OATPs. The behavior of
cellular uptake was further quantified using flow cytometry (Figure 4 C). The mean
fluorescence intensity of PC-3 cells incubated with NIR-INH and MHI-148 at 37 °C was
much higher than that treated at 4 °C and pretreated BSP (Figure 4 D). Taken together,
cellular uptake of MHI-148 and NIR-INH is an energy-dependent process and mediated by
OATPs in PC-3 cancer cells. NIR-INH and MHI-148 accumulated in the PC-3 cellular
mitochondria by exclusively colocalized with MitoTracker.
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Figure 4. Subcellular localization and cell uptake of MHI-148 and NIR-INH. (A) CLSM
images of PC-3 cells co-stained with MHI-148/NIR-INH and MitoTracker. (B) CLSM images
of PC-3 cells treated with 20 μM MHI-148 or MHI-148 in different conditions. (C) Cellular
uptake of MHI-148 and NIR-INH measured by flow cytometry. (D) The mean fluorescence
intensity of PC-3 cells incubated with NIR-INH and MHI-148.
3.5. In vivo biodistribution
In vivo distribution of INH, MHI-148 and NIR-INH was examined in PC-3 tumor
bearing nude mice, and visualized using IVIS imaging. The preparation was administrated
intravenously at a dose of 2.5 μM kg-1. As shown in Figure 5 A and B, the mean fluorescence
intensity of tumors was stronger than other organs except lung at 48 h. The fluorescence
intensity in tumors of NIR-INH group showed no significant difference compared with that of
MHI-148. The selective tumor accumulation supports the use of NIR-INH for tumor
localization, and thus, it could provide a precise imaging-guided cancer therapy. This
conjugation with a proper modification of one carboxyl group of MHI-148 did not change the
lipophilic cationic property of its hepatemethine core and keep the fluorescence and tumor-
targeting functions. It should be noted that not all cyanine dyes possess tumor-specific
targeting function in spite of their similar structures. Only heptamethine carbocyanine dyes
having heptamethine core with lipophilic cationic property are able to accumulate in tumor
cells [21, 28-30].
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Figure 5. In vivo distribution in nude mice bearing PC-3 tumors. (A) NIR fluorescence images
of organs and tumors at 48 h post-injection of saline, INH, MHI-148, and NIR-INH. (B) NIR
fluorescence intensity of organs and tumors treated with MHI-148 and NIR-INH.
3.6. In vivo tumoricidal activities of NIR-INH
Based on the favorable anti-tumor effect of NIR-INH in vitro, we further investigated the
in vivo anti-tumor activities of NIR-INH in tumor xenograft models bearing PC-3 tumors.
Saline, INH, MHI-148, or NIR-INH was injected intravenously into mice every 3 days, with a
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total of 5 injections. As shown in Figure 6 A and B, MHI-148 demonstrated a moderate anti-
tumor effect of delayed tumor progression, as compared to the saline and INH groups. This
may due to accumulation of MHI-148 in the mitochondria of PC-3 cells, and this could induce
cancer cell apoptosis by disrupting mitochondrial activities [19]. We also found that mice
treated with NIR-INH displayed a significant decrease in tumor volume. Tumor burden and
tumor inhibition rate were calculated by excising and weighing tumor tissues. NIR-INH
demonstrated the highest levels of tumor suppression. It had a TIR of 80.39%, which is 1.72-
and 3.86-fold higher than MHI-148 and INH, respectively (Figure 6 C). Serum prostate-
specific antigen (PSA) levels are utilized extensively as a biomarker for PCa diagnose,
evaluation of PCa progression, and evaluation of response to systemic therapies [5, 6]. PSA
levels in mice were analyzed on day 14, and NIR-INH treated mice had a significant
reduction in PSA levels (Figure 6 D). MAOA inhibitory activities were evaluated in serum
after INH, NIR-INH, and MHI-148 treatment by a ELISA kit (Cusabio Biotech, China). As
shown in Figure 6 E, NIR-INH showed a significant reduction in MAOA level compared with
INH. The result indicated that NIR-INH may possess higher MAOA inhibition efficacy than
INH with the terminal hydrazide group conjugated with MHI-148. The marked antitumor
activity of NIR-INH may be attributed to the preferential accumulation of NIR-INH mediated
by OATPs and the increased MAOA inhibitory activity derived from INH N’-acylation [25-
27].
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Figure 6. In vivo antitumor effects of INH, MHI-148, and NIR-INH in PC-3 tumor xenografts.
(A) Tumor volume following intravenous administration of saline, INH, MHI-148, and NIR-
INH (n=5). (B) Morphology of dissected tumors from PC-3 tumor-bearing mice following
intravenous administration of saline, INH, MHI-148, and NIR-INH (n=5). (C) Tumor burden
(weight of tumors divided by the average body weights of mice) and tumor inhibition rate
(TIR) of mice administered with saline, INH, MHI-148, and NIR-INH. (D) Serum PSA levels
in PC-3 tumor-bearing mice analyzed after the treatment using ELISA. (E) Serum MAOA
levels in PC-3 tumor-bearing mice determined after the treatment by ELISA. (F) TUNEL
assay images of tumor specimens treated with saline, INH, MHI-148, and NIR-INH. The data
are presented as mean±SD, P values: **P < 0.01, *** P < 0.001.
The TUNEL assay was used to evaluate if apoptosis is induced in tumor cells by INH,
MHI-148, and NIR-INH treatment (Figure 6 F). A robust and significant increase in cell
apoptosis was observed in tumor samples treated with NIR-INH, as compared to both INH
and MHI-148 treatment. Additionally, there was no significant change observed in body
weight or in the hematological parameters between treated and control groups (Figure 7 A, B).
Furthermore, no noticeable histological abnormalities were observed in H&E staining of
major organ sections. This confirms that the NIR-INH conjugate has potent antitumor activity
and low treatment-induced toxicity (Figure 8). Xu et al. had previously shown that MHI-148
(IR-808) and its derivative (IR-808DB) not only possess tumor-targeting and imaging
functions, they also have remarkable photodynamic therapy (PDT) activity [17, 18]. Thus, the
antitumor activity of NIR-INH combined with PDT is under investigation to further improve
PCa treatment.
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Figure 7. Body weight and hematological parameters. (A) Body weight changes following
treatment of saline, INH, MHI-148, and NIR-INH (n=5). (B) Serum markers of hepatic and
renal function in mice bearing PC-3 tumor xenografts after treatment with saline, INH, MHI-
148, and NIR-INH (n=5).
Figure 8. CLSM images of H&E staining in organs after the treatment with saline, INH, MHI-
148, and NIR-INH.
4. Conclusion
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In this study, we reported an effective tumor-targeting NIRF dye−MAOA inhibitor
conjugate, termed NIR-INH, is a promising imaging and targeting agent for the diagnosis and
therapy of human PCa. The accumulation of NIR-INH in the mitochondria of PC-3 cells is
mediated by OATPs. The anti-tumor activity, mainly derived through MAOA inhibition and
mitochondria targeting, makes it a valuable agent for the therapy and diagnosis of human PCa.
Author information
Corresponding Authors
*Phone: +86-24-23986325; E-mail: wangyongjun@syphu.edu.cn
*Phone: +86-24-23986428; E-mail: wangduncn@hotmail.com
*Phone: +86-24-23986321; E-mail: hezhonggui@vip.163.com
Acknowledgments
This research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.
Appendix A. Supplementary data
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