Design, Synthesis, and Biological Evaluation of Mitochondria-Targeted Flavone-Naphthalimide-Polyamine Conjugates with Antimetastatic Activity

Article abstract of DOI:10.1021/acs.jmedchem.6b01846

Approximately 90% of cancer-associated deaths result from disseminated tumors, indicating the ineffectiveness of current therapies and the imperative need of antimetastatic drugs. A novel pharmacophore with flavonoid and naphthalimide moieties was constructed by using a fragment-based drug design and a series of eight flavone-naphthalimide-polyamine conjugates were synthesized. In vitro evaluation revealed that compound 6c with a homospermidine motif displayed better cell selectivity between cancerous and normal liver cells than amonafide did. The in vivo assays on two hepatocellular carcinoma (HCC) models verified that 6c potently suppressed pulmonary metastasis with improved organ indexes compared to amonafide. Various experiments showed that 6c as a potential fluorescent chemical probe could target the mitochondria. Preliminary investigation into the mechanism of action of 6c indicated that it might harness a polyamine transporter for cell entrance, localize in the mitochondria, selectively cause reactive oxygen species (ROS) overproduction in hepatoma cells instead of normal liver cells, and finally lead to HCC cell apoptosis and migration inhibition via multiple ROS-mediated signaling pathways.

Full text of DOI:10.1021/acs.jmedchem.6b01846

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Article
Design, Synthesis and Biological Evaluation of Mitochondria-Targeted
Flavone-naphthalimide-polyamine Conjugates with Antimetastatic Activity
Fujun Dai, Qian Li, Yuxia Wang, Chaochao Ge, Chenyang Feng,
Songqiang Xie, Haoying He, Xiaojuan Xu, and Chaojie Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01846 • Publication Date (Web): 08 Feb 2017
Downloaded from http://pubs.acs.org on February 9, 2017
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Design, Synthesis and Biological Evaluation of
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MitochondriaꢀTargeted
Flavoneꢀnaphthalimideꢀ
polyamine Conjugates with Antimetastatic Activity
Fujun Dai, † Qian Li, † Yuxia Wang,* ‡ Chaochao Ge, † Chenyang Feng,§ Songqiang Xie, *§
Haoying He,§ Xiaojuan Xu, § Chaojie, Wang*†
†Key Laboratory of Natural Medicine and ImmunoꢀEngineering
‡College of Chemistry and Chemical Engineering
§Pharmaceutical College, Henan University, Kaifeng 475004, Henan, China.
KEYWORDS
Flavoneꢀnaphthalimideꢀpolyamine conjugate
Mitochondria
Reactive oxygen species
Apoptosis
Metastasis
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ABSTRACT
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Approximately 90% of cancerꢀassociated deaths result from disseminated tumors, indicating the
ineffectiveness of current therapies and the imperative need of antimetastatic drugs. A novel
pharmacophore with flavonoid and naphthalimide moieties was constructed by using a fragmentꢀ
based drug design and a series of eight flavoneꢀnaphthalimideꢀpolyamine conjugates were
synthesized. In vitro evaluation revealed that compound 6c with a homospermidine motif
displayed better cell selectivity between cancerous and normal liver cells than amonafide did.
The in vivo assays on two hepatocellular carcinoma (HCC) models verified that 6c potently
suppressed pulmonary metastasis with improved organ indexes compared to amonafide. Various
experiments showed that 6c as a potential fluorescent chemical probe could target the
mitochondria. Preliminary investigation into the mechanism of action of 6c indicated that it
might harness a polyamine transporter for cell entrance, localize in the mitochondria, selectively
cause reactive oxygen species (ROS) overproduction in hepatoma cells instead of normal liver
cells, and finally lead to HCC cell apoptosis and migration inhibition via multiple ROSꢀmediated
signaling pathways.
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1. INTRODUCTION
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Hepatocellular carcinoma (HCC) ranks as the sixth most prevalent malignancy and the third
cause of cancerꢀassociated mortality worldwide. The unsatisfactory longꢀterm survival of HCC
patients is mainly due to metastasis and recurrence after various standard therapies.1, 2 Indeed,
tumor metastasis is a major obstacle in present chemotherapy and is responsible for
approximately 90% of cancerꢀpatient deaths. This indicates that current anticancer agents
including conventional cytotoxic compounds and rationally designed targeted drugs can cure
wellꢀdefined primary tumors, but have limited impacts on disseminated cells.3, 4 Moreover, some
clinically used antitumor drugs trigger tumors to spread from the primary tumor mass to distant
sites.5 Thus, the design of novel antimetastatic therapeutic agents and relevant mechanism study
represent areas of urgent attention.
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Naphthalimides as versatile functional compounds have received durable attention for both
their promising antitumor activities and photophysical characters.6ꢀ8 For example, amonafide
(Figure 1) has reached clinical trials for both primary and metastatic tumors.9ꢀ11 However, most
recent naphthalimideꢀbased drug designs focus on treating primary tumors rather than metastatic
ones. Though naphthalimides are known as DNA intercalators, some fluorescent naphthalimides,
used as imaging agents, may alternatively localize in other subcellular organelles (such as
lysosomes and mitochondria) instead of the nucleus.6, 12ꢀ18 This implies that some naphthalimide
derivatives may have cellular targets distinct from DNA.
The native polyamines existing in almost every living cell have complex biological properties.
Natural and synthetic polyamines, attractive as ligands, have been investigated to couple with
diverse functional groups.19ꢀ23 Modification with a polyamine may endow the parent drug with
some intriguing biological features. Several studies have reported that the presence of a
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polyamine might be beneficial in dealing with some major issues in current tumor treatment.
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Indeed,
F14512
[(2ꢀ{3ꢀ[4ꢀ3ꢀaminopropylamino)ꢀbutylamino]ꢀpropylamino}ꢀ4′ꢀdemethylꢀ
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epipodophyllotoxinꢀ4ꢀyl) acetamide], one of the most promising polyamine conjugates with a
spermineꢀvectorized epipodophyllotoxin core, is currently in clinical trials for the treatment of
refractory/relapsing acute myeloid leukemia.24ꢀ27 The Phanstiel group recently found that the
polyamine conjugate could inhibit the in vivo metastasis of human L3.6pl pancreatic cancer cells
to the liver.28
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In our search for polyamineꢀrelated antitumor agents, polyamine conjugates of naphthalimide,
naphthalene diimide, and flavonoid with a naphthalene motif (Figure 1) displayed potential
antimetastatic activities.29ꢀ31 However, their lack of definite cellular localization led us to develop
a new structure with dual functions for bioimaging and antimetastatic activity. Guided by the
fragmentꢀbased approach, we have described the construction of a novel flavoneꢀnaphthalimideꢀ
polyamine conjugate, which combines the respective photophysical and anticancer properties of
three structural components (Figure 1). The target compound 6c could inhibit both the primary
liver tumor growth and hepatoma pulmonary metastasis in vitro and in vivo. The mechanistic
experiments revealed that 6c could enter cells via a polyamine transporter (PAT), localize in the
mitochondria, selectively elevate the reactive oxygen species (ROS) level of cancerous liver
cells, induce apoptosis, and inhibit tumor metastasis.
2. RESULTS AND DISCUSSION
2.1. Chemistry. Eight polyamine conjugates with flavoneꢀnaphthalimide as a novel
pharmacophore were synthesized according to Scheme 1. 5ꢀAcenaphthenecarboxaldehyde (2)
was first prepared by the conventional Vilsmeier reaction with acenaphthene (1) as the starting
material in 85% yield. Then, the flavone with a naphthalene fragment (3), obtained from the
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reaction of 2 with 2ꢀhydroxyacetophenone, was oxidized by K2Cr2O7 to yield the anhydride (4)
in 83% yield. The condensation of 4 with corresponding amines/polyamines (R1NH2, Figure 2),
and the subsequent neutralization or Bocꢀgroup removal with 4 M HCl resulted in the target
compounds 6aꢀ6h in the form of hydrochloride salts in 83ꢀ86% yield. The structures of all the
new compounds were confirmed via 1H NMR, HRMS, and elemental analysis.
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2.2. In vitro biological evaluation. In vitro assays were performed to evaluate the inhibitory
effect of the target compounds on two tumor cell lines after treatment for 48 h, SMMCꢀ7721
(hepatoma cell line), and HepG2 (hepatoma cell line), and one normal hepatocyte cell line QSGꢀ
7701 by using traditional 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT)
tests. Amonafide was selected as the reference drug. The biological results of the target
compounds are shown in Table 1. Compounds 6aꢀ6d bearing linear monoꢀ, diꢀ, and triꢀamine
motifs showed better activity than 6eꢀ6h with branched or cyclic amino groups against HepG2,
and SMMCꢀ7721 cell lines. Moreover, the human normal hepatocyte cells were not sensitive to
6b and 6c relative to amonafide. These results indicated that the amino side chain of flavoneꢀ
naphthalimideꢀpolyamine conjugates played an important part in their inhibitive behaviors. The
inhibitory effect of 6c with a synthetic homospermidine motif on hepatoma cells was confirmed
via colony formation assays (Figure 3A). More cellꢀselectivity experiments supported that QSGꢀ
7701 cells did not significantly respond to 6c compared to hepatoma cells even after 72 h (Figure
3B).
Migration is a key step during metastasis. We previously found that flavonoidꢀpolyamine
conjugates could inhibit tumor metastasis moderately31 and naphthaleneꢀdiimideꢀpolyamine
conjugates could inhibit tumor metastasis potently in vivo.30 Therefore, transwell migration
assays were conducted to determine whether 6c could prevent the migration of hepatoma cells.
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As illustrated in Figure 3C, the presence of 6c markedly attenuated the migration of HepG2 and
SMMCꢀ7721 cells in a doseꢀdependent manner.
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2.3. Inhibition of tumor growth and metastasis in vivo. To evaluate the antitumor effect of 6c
in vivo, we used two HCC animal models including a primary tumor growth model and a
pulmonary metastasis model. Mice bearing hepatoma xenografts were treated with 6c (3 mg/kg),
amonafide (5 mg/kg, positive control), and normal saline (negative control) once every day for 7
days by tail vein. The tumor suppression of 6c (60.4%) was slightly better than that of amonafide
(48.1%) (Figure 4A and 4B). Meanwhile, the variations of organ weight indexes implied that 6c
had no obvious pathological changes in the experiments of toxicological profile in vivo (Figure
4C), which was consistent with the effect of 6c on QSGꢀ7701 cells.
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Metastasis is the main factor affecting mortality in cancer patients, and the lung is one of the
most common sites for extrahepatic metastasis.32 To assess whether 6c was effective against
pulmonary metastasis in vivo, mice were injected with cancer cells via the tail vein. After 6c
treatment (3 mg/kg), significantly reduced numbers of tumor metastatic nodules (77.9%) were
observed. Moreover, the inhibitory effect of 6c on metastasis was better than that of amonafide
(5 mg/kg, 41.4%) (Figure 4D). In addition, the size of metastatic nodules examined
by hematoxylinꢀeosin (H&E) staining was obviously smaller in 6cꢀtreated group than the
negativeꢀcontrol group (Figure 4E). As mentioned above, many clinically used drugs are
effective in curing primary tumors, but ineffective in inhibiting tumor metastasis. Indeed,
mitoxantrone, a clinical drug for HCC, could potently regress the primary tumor growth (73.4%)
compared to the moderate inhibition of pulmonary metastasis (49.2%) in similar assays in vivo.31
Our results from both in vitro and in vivo experiments illustrate that 6c was more powerful in
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preventing tumor metastasis than inhibiting primary tumor growth. This evidence supports the
hypothesis that polyamine conjugates may serve as potential antimetastatic agents.
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2.4. Cellular uptake and localization. The presence of naphthalimide moiety in 6c guaranteed
the expected fluorescent properties with a maximum fluorescent emission wavelength at 430 nm.
To evaluate the potential property of 6c for biological imaging in live cells, high content screen
(HCS) system was used, and blue fluorescence of 6cꢀtreated hepatoma cells was observed in a
doseꢀdependent (Figure 5A) and timeꢀdependent manner (Figure 5B and 5C). These results
showed that 6c could be taken up by cells efficiently.
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Cancer cells often possess an active polyamine transport (PAT) system to import native and
structurally modified polyamines.33, 34 To detect whether the cellular uptake of 6c was dependent
on PAT, a natural PAT substrate, spermidine (Spd) was used to compete with the potential PATꢀ
selective polyamine conjugates. Coꢀincubation of 6c with Spd at a concentration that did not
affect cell viability decreased the fluorescence intensity of 6c in cells compared to treatment with
6c alone (Figure 6A and 6B). The attenuated concentration of 6c with Spd subsequently led to
the reduced inhibitory effect of 6c on hepatoma cells (Figure 6C). In addition, the expression of
two critical enzymes in polyamine metabolism,35 ornithine decarboxylase (ODC) and
spermidine/spermine N1ꢀacetyltransferase (SSAT), was not affected by 6c (Figure 6D), which
was in accordance with the slight change in the polyamine pool levels including putrescine,
spermidine, and spermine levels (Figure 6E). Therefore, 6c might penetrate the cell membrane at
least partially via PAT and did not influence polyamine metabolism.
To visualize the cellular localization of 6c, various organelleꢀspecific fluorescent dyes were
used. In Figure 7A and 7B, we did not observe the localization of 6c in the membrane and the
nucleus as determined by using the membraneꢀspecific dye DiO and nucleusꢀspecific dye
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propidium iodide (PI), respectively. Confocal microscopy was used to detect the subcellular
localization of 6c with other organelleꢀspecific labels, such as Lysoꢀtracker for lysosome, ERꢀ
tracker for endoplasmic reticulum (ER), Golgiꢀtracker for Golgi, and Mitoꢀtracker for
mitochondria. In 6cꢀtreated hepatoma cells, we did not find that 6c localized in the lysosome,
ER, or Golgi (Figure 7C). Inspection of merged images suggested that 6c might selectively
localize within the mitochondria (Figure 7D). To confirm the presence of 6c in mitochondria,
mitochondria were isolated from 6cꢀtreated cells. The blue fluorescence of mitochondria was
observed in 6cꢀtreated group but not the control group (Figure 7E). Therefore, 6c might serve as
a novel fluorogenic probe for mitochondria.
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2.5. Induction of apoptosis. Localization to the mitochondria insinuated that 6c might exert its
inhibitory activity through mitochondria. Therefore, the mitochondrial membrane potential
(MMP, ꢁψm) of HepG2 cells stained with Rh123 or JCꢀ1 was tested by flow cytometry. Rh123
is a cationic dye with green fluorescence. It is sequestered in mitochondria based on the highly
negative MMP (ꢁψm) and released upon mitochondrial depolarization. JCꢀ1 is a mitochondriaꢀ
selective aggregate dye. At high membrane potentials, JCꢀ1 forms “Jꢀaggregates” displaying red
fluorescence. The fluorescence variations of Rh123 and JCꢀ1 in 6cꢀtreated cells implied that 6c
treatment evidently altered the mitochondria function and mitochondrial membrane potential
(Figure 8A).
Mitochondria participate in the induction of apoptosis, and loss of ꢁψm is an early event in
apoptotic process.36, 37 Apoptotic morphology was first studied by measuring acridine orange
(AO) and ethidium bromide (EB) staining via HCS. Viable cells stained by AO have bright green
fluorescence, while apoptotic cells stained by AO and EB show bright orange fluorescence. The
reduced fluorescence intensity of AO and elevated fluorescence intensity of EB indicated that 6c
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induced apoptosis in a doseꢀdependent manner (Figure 8B). The 6cꢀtriggered apoptosis was
confirmed via flow cytometry using Annexin V/PI double staining.38, 39 The number of early
apoptotic and late apoptotic cells was evenly distributed in the total number of apoptotic cells,
suggesting that 6c could induce both early and late apoptosis (Figure 8C). Caspase 3 is one of the
key proteases in apoptosis. Therefore, the activation of caspase 3 was measured via
immunofluorescence, by using a primary antibody against the cleaved portions of caspase 3.
Indeed, 6c treatment resulted in the upregulation of cleaved caspase 3 in HepG2 cells in a doseꢀ
dependent manner (Figure 8D). Similar results were obtained via western blot (Figure 8E).
Interestingly, the presence of 6c raised the expression of cleaved caspase 3 in HepG2 cells but
not in QSGꢀ7701 cells (Figure 8E). This is in accordance with the previously described MTT
assays (Table 1 and Figure 3B) in which 6c showed lower cytotoxicity for QSGꢀ7701 cells
relative to hepatoma cells.
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2.6. Role of ROS in apoptosis and migration. Mitochondria are the major source of ROS that
are closely connected with apoptosis.40 Intracellular ROS generation was thus evaluated via 2',7'ꢀ
dichlorodihydrofluorescein diacetate (DCFHꢀDA) staining. In Figure 9A, 6c treatment increased
the mean fluorescence intensity by nearly 2.6ꢀ and 2.2ꢀ fold in HepG2 and SMMCꢀ7721 cells,
respectively. However, the fluorescence intensity in QSGꢀ7701 cells was not sensitive to 6c
•−
treatment (Figure 9B). ROS include hydrogen peroxide (H2O2) and superoxide anion (O2 ). The
•−
content of O2 in 6cꢀtreated cells was obviously increased while 6c treatment slightly
upregulated the level of H2O2 (Figure 9C). Alteration of the oxidant/antioxidant balance triggers
apoptosis, and glutathione (GSH) as an endogenous antioxidant against ROS has important
functions.41ꢀ43 The partial depletion of GSH content by 6c could also increase the cellular ROS
generation (Figure 9C). To explore the effects of ROS scavengers on 6cꢀinduced ROS
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generation, NꢀacetylꢀLꢀcysteine (NAC), alphaꢀtocopherol (TOC), or catechin (CAT) were
used.44, 45 At the specified concentration, these three ROS scavengers did not show obvious
cytotoxicity on hepatoma cells (Figure 9D). Pretreatment with these ROS scavengers evidently
reduced the 6cꢀelevated fluorescence intensity of DCFHꢀDA (Figure 9E).
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To probe the role of ROS in 6cꢀinduced apoptosis, hepatoma cells were pretreated with ROS
scavengers before the addition of 6c. The higher cell viability in ROS scavengersꢀtreated cells
indicated that the presence of the three ROS scavengers obviously protected the cells from the
attack of 6c (Figure 10A), indicating the critical function of ROS in the cytotoxicity of 6c.
Meanwhile, ROS scavengers treatment evidently reduced 6cꢀinduced the expression of cleaved
caspase 3 and poly ADPꢀribose polymerase (PARP) which is susceptible to caspase 3 (Figure
10B). This implied that ROS regulated the expression of caspase 3 and PARP in the signaling
pathway. In addition, ROS scavenger pretreatment elevated the migration ability of 6cꢀtreated
hepatoma cells (Figure 10C), suggesting that 6c might inhibit cell migration via ROS. p53, as a
tumor suppressor, plays an important role in the progression of a tumor, including cancer cell
migration and invasion.46 To detect the effect of 6c on p53 expression, immunofluorescence
assays and western blots were carried out. The expression of p53 induced by 6c was obviously
elevated in a doseꢀdependent manner compared to the negative group (Figure 10D and 10E).
There is a versatile partnership between p53 and ROS. 42, 47 NACꢀpretreatment partially reduced
the level of p53 expression (Figure 10E), supporting the association of p53 with 6cꢀinduced ROS
generation. All these findings showed that the ROS production is not only a crucial event in 6cꢀ
induced apoptosis and migration inhibition of hepatoma cells, but also the main reason for the
lower cytotoxicity of 6c to normal liver cells.
3. CONCLUSION
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Collectively, we have described the construction of a novel flavoneꢀnaphthalimide scaffold and
the synthesis of a series of flavoneꢀnaphthalimideꢀpolyamine conjugates. The in vitro SAR
studies led to the discovery of a small molecule 6c with a linear homospermidine motif, which
showed antitumor activity similar to that of amonafide, but better cell selectivity between
cancerous and normal liver cells. Further in vivo assays on two HCC models showed that 6c
could potently prevent pulmonary metastasis with fewer side effects compared to amonafide.
Extensive assays supported that 6c might serve as a fluorescent probe for mitochondria. The
mechanistic study clearly showed the critical role of ROS in 6cꢀinitiated apoptosis and metastasis
inhibition. 6c might enter targeted cells via a polyamine transporter, and then preferentially
localize in the mitochondria and selectively generate ROS in hepatoma cells. The generated ROS
subsequently resulted in cell apoptosis and migration inhibition. Thus, 6c with the dual features
of bioimaging and antimetastatic properties is a valuable lead compound for future development
of antimetastatic agents.
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4. EXPERIMENTAL PROTOCOLS
4.1. Chemistry. All solvents and reagents were purchased from suppliers and used without
further purification. All 1H NMR spectra were recorded on a Bruker AVꢀ400 model spectrometer
1
in D2O or CDCl3, Chemical shifts for H NMR spectra are reported in parts per million to
residual solvent protons. High resolution mass spectrometry was performed on a QꢀTOF with
ESI ionisation. ESIꢀMS spectrum (low resolution) was recorded on an ESQUIREꢀLC mass
spectrometer. The target compounds with the purity being higher than 95% were analyzed using
combustion analysis, performed on a Gmbe VarioEL elemental instrument, and results were
within 0.4% of theoretical values. Diverse amine/polyamine motifs (Figure 2) were prepared by
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previous approaches. 30
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4.1.1. Synthesis of Intermediate 3. To a solution of acenaphthene 1 (9 mmol, 1.38 g) in POCl3
(1.35 mL) was added DMF (1.35 mL) dropwise, the mixture was stirred at 90~95℃ for 3 h.
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Then the reaction mixture was poured into ice water (27 mL). After filtration, the brown solid
was dried to give the aldehyde 2 with a crude yield of 85%, which was used without further
purification.
To a mixture of 2ꢀacetylphenol (7.5 mmol, 1.02g) and compound 2 (9.95 mmol, 1.81 g) in
EtOH (5.5 mL) was added 60% KOH/EtOH (12 mL), the mixture was then left at room
temperature for about 48 h with occasional shaking. After 48 h, it was poured into ice water and
the hydrochloric acid was added to adjust the solution to pH=3~4. An orange solid was obtained
after filtration. Without further purification, the orange solid were dissolved in DMSO (13.5
mL), then, a catalytic amount of I2 was added and stirred for about 30–60 min at 140°C. After
cooling down to room temperature, ice water (100 mL) was added. Then a small amount of
Na2S2O3 was added and stirred for 30 min to remove I2. After filtration, the crude intermediate
was purified by column chromatography with petroleum ether/acetone (8:1, v/v) as elution
solvent to give the flavone 3 as a pale yellow solid with a yield of 80%.
2ꢀ(1,2ꢀdihydroacenaphthylenꢀ5ꢀyl)ꢀ4Hꢀchromenꢀ4ꢀone (3). 1HNMR (CDCl3): δ 8.30(d,
J=10.64 Hz, 1H, ArꢀH), 8.01(d, J=10.88 Hz, 1H, ArꢀH); 7.85(d, J=9.24 Hz, 1H, ArꢀH); 7.69ꢀ
7.75(m, 1H, ArꢀH); 7.54ꢀ7.59(m, 2H, ArꢀH); 7.38ꢀ7.48(m, 3H, ArꢀH); 6.77(s, 1H, ArꢀH); 3.47(s,
4H, 2×CH2). ESIꢀMS m/z: 299.3[M+H]+.
1.2. Synthesis of Target Compounds. A mixture of compound 3 (2.5 mmol, 1.5g), potassium
dichromate (3.40 mmol, 4g), and CH3COOH (22 mL) were stirred at 90~100°C for 2 h. After
cooling down to room temperature, the reaction mixture was poured into 250 mL ice water. After
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filtration, the residue was dried to give crude intermediate anhydride 4 as milky white solid with
a yield of 83%, which was used for next step without further purification.
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A mixture of the compound 4 and diverse amine/polyamine (in a 1:1 molar ratio) dissolved in
EtOH was heated at refluxing temperature and monitored by TLC. After completion, the reaction
mixture was cooled to room temperature and concentrated under vacuum to give an oily residue.
After purification by column chromatography with dichloromethane/methanol (100:0.5ꢀ100:3,
v/v) as elution solvent, the obtained 5aꢀ5h was dissolved in CH3CH2OH. 4 M HCl was added at
0°C, and then the mixture was stirred at room temperature overnight until a great amount of
precipitate was generated. The filtered cake was washed by anhydrous CH3CH2OH, dried to give
the target compound 6aꢀ6h as a hydrochloride salt.
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2ꢀ{2ꢀ[(4ꢀbutyl)–aminobutyl]1Hꢀbenz[de]isoquinolineꢀ1,3(2H)ꢀdione}ꢀ6ꢀ4Hꢀchromeneꢀ4ꢀone
hydrochloride (6a). Yield 86%, white solid. 1H NMR(400 MHz, D2O) δ:7.76ꢀ7.85(m, 3H, ArꢀH);
7.46(d, J=7.56 Hz, 1H, ArꢀH); 7.31ꢀ7.36(m, 2H, ArꢀH); 7.14ꢀ7.17(m, H, ArꢀH); 7.07(t, J=7.44
Hz, 1H, ArꢀH); 6.83(d, J=8.2 Hz, 1H, ArꢀH); 5.82(s,1H, ArꢀH); 3.88(t, J=6.48 Hz, 2H, 1×Nꢀ
CH2); 3.05ꢀ3.13(m, 4H, 2×NꢀCH2); 1.74ꢀ1.78(m, 2H, 1×CH2); 1.64ꢀ1.73(m, 4H, 2×CH2); 1.36ꢀ
1.45(m, 2H, 1×CH2); 0.95(t, 3H, J=7.36Hz, 1×CH3). HRMS (TOF MS ESI, [M–HCl+H]+): m/z
calculated for C29H29N2O4: 469.2127, found: 469.2124. Anal.calcd for C29H29ClN2O4·0.6H2O: C
67.53%, H 5.90%, N 5.43%; found: C 67.36%, H 5.90%, N 5.49%.
2ꢀ{2ꢀ[3ꢀ(4ꢀaminobutyl)–aminopropyl]1Hꢀbenz[de]isoquinolineꢀ1,3(2H)ꢀdione}ꢀ6ꢀ4Hꢀ
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chromeneꢀ4ꢀone dihydrochloride (6b). Yield 85%, white solid. H NMR (400 MHz, D2O) δ:
7.73ꢀ7.77(m, 3H, ArꢀH); 7.39ꢀ7.42(m, 1H, ArꢀH); 7.25ꢀ7.29(m, 2H, ArꢀH); 7.01ꢀ7.05(m, 2H, Arꢀ
H); 6.70ꢀ6.73(m, 1H, ArꢀH), 5.63(d, J=4.68 Hz,1H, ArꢀH); 3.98(s, 2H, 1×NꢀCH2); 3.15ꢀ3.22(m,
4H, 2×NꢀCH2); 3.10(t, J=7.12 Hz, 2H, 1×NꢀCH2); 2.03~2.07(m, 2H, 1×CH2); 1.76ꢀ1.90(m, 4H,
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2×CH2). HRMS(TOF MS ESI, [M–2HCl+H]+): m/z calculated for C28H28N3O4: 470.2080,
found: 470.2066. Anal.calcd for C28H29Cl2N3O4·1.6H2O: C 58.87%, H 5.68%, N 7.36%; found:
C 58.97%, H 5.57%, N 7.45%.
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2ꢀ{2ꢀ[4ꢀ(4ꢀaminobutyl)–aminobutyl]1Hꢀbenz[de]isoquinolineꢀ1,3(2H)ꢀdione}ꢀ6ꢀ4Hꢀ
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chromeneꢀ4ꢀone dihydrochloride (6c). Yield 85%, white solid. H NMR (400 MHz, D2O) δ:
7.89ꢀ7.92(m, 2H, ArꢀH); 7.81(d, J=8.0 Hz,1H, ArꢀH); 7.52(d, J=8.0 Hz, 1H, ArꢀH); 7.31ꢀ7.40(m,
2H, ArꢀH); 7.18(d, J=4.0 Hz, 1H, ArꢀH), 7.07(t, J=8.0 Hz, 1H, ArꢀH); 6.87(d, J=12.00 Hz, 1H,
ArꢀH); 5.91(s, 1H, ArꢀH); 3.89(s, 2H, 1×NꢀCH2); 3.05~3.10(m, 4H, 2×NꢀCH2); 2.99(t, J=6.00
Hz, 2H, 1×NꢀCH2); 1.65~1.73(m, 8H, 4×CH2). HRMS(TOF MS ESI, [M–2HCl+H]+): m/z
calculated for C29H30N3O4 : 484.2236, found: 484.2224. Anal.calcd for C29H31Cl2N3O4·3.35H2O:
C 56.47%, H 6.16%, N 6.81%; found: C 56.86%, H 6.24%, N 6.43%.
2ꢀ{2ꢀ[3ꢀ(3ꢀ(3ꢀaminopropyl)aminopropyl)ꢀaminopropyl]ꢀamino}1Hꢀbenz[de]isoquinolineꢀ
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1,3(2H)ꢀdione}ꢀ6ꢀ4Hꢀ chromeneꢀ4ꢀone trihydrochloride (6d). Yield 83%, white solid. H NMR
(400 MHz, D2O) δ: 7.96ꢀ8.05(m, 3H, ArꢀH); 7.64(d, J=7.68 Hz,1H, ArꢀH); 7.50(t, J=7.92
Hz,1H, ArꢀH); 7.39ꢀ7.44(m, 1H, ArꢀH); 7.30(d, J=7.72 Hz, 1H, ArꢀH), 7.18(t, J=7.6 Hz,1H, Arꢀ
H); 6.94(d, J=8.32 Hz, 1H, ArꢀH); 5.99(s,1H, ArꢀH); 4.09(t, J=6.68 Hz ，2H, 1×NꢀCH2);
3.20~3.26(m, 8H, 4×NꢀCH2); 3.13(t, J=7.86 Hz, 2H, 1×NꢀCH2); 2.08ꢀ2.25(m, 6H, 3×CH2).
HRMS(TOF MS ESI, [M–3HCl+H]+): m/z calculated for C30H33N4O4:513.2502, found:
513.2476. Anal.calcd for C30H35Cl3N4O4·1.0H2O: C 56.30%, H 5.83%, N 8.75%; found: C
56.03%, H 5.84 %, N 8.88%.
2ꢀ{2ꢀ[(3ꢀdibutyl)–aminopropyl]1Hꢀbenz[de]isoquinolineꢀ1,3(2H)ꢀdione}ꢀ6ꢀ4Hꢀchromeneꢀ4ꢀ
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one hydrochloride (6e). Yield 84%, white solid. H NMR (400 MHz, D2O) δ: 7.82ꢀ7.88(m, 2H,
ArꢀH); 7.75ꢀ7.77(m,1H, ArꢀH); 7.49(d, J=7.08 Hz, 1H, ArꢀH); 7.39(t, J=7.62 Hz,1H, ArꢀH);
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7.28ꢀ7.32(m, 1H, ArꢀH); 7.17(d, J=7.88 Hz,1H, ArꢀH); 7.11(t, J=7.28 Hz,1H, ArꢀH); 6.89(d,
J=8.32 Hz, 1H, ArꢀH); 5.68(s, 1H, ArꢀH); 3.99(t, 1H, J=5.88 Hz, 1×NꢀCH2); 3.28(t, J=7.8 Hz,
2H, 1×NꢀCH2); 3.21(t, J=8.24 Hz, 4H, 2×NꢀCH2); 2.06~2.13(m, 2H, 1×CH2); 1.68~1.77(m, 4H,
2×CH2); 1.36~1.46(m, 4H, 2×CH2); 0.95(t, 6H, J=7.38 Hz, 2×CH3). HRMS(TOF MS ESI, [Mꢀ
HCl+H]+ ): m/z calculated for C32H35N2O4: 511.2597, found: 511.2666. Anal.calcd for
C32H35ClN2O4·1.0H2O: C 68.01%, H 6.60%, N 4.96%; found: C 67.73%, H 6.63%, N 4.89%.
2ꢀ{2ꢀ[(3ꢀpiperazinꢀ1ꢀyl)–propyl]1Hꢀbenz[de]isoquinolineꢀ1,3(2H)ꢀdione}ꢀ6ꢀ4Hꢀchromeneꢀ4ꢀ
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one dihydrochloride (6f). Yield 86%, white solid. H NMR (400 MHz, D2O) δ: 7.83~7.89(m,
3H, ArꢀH); 7.50ꢀ7.52(m,1H, ArꢀH); 7.33ꢀ7.39(m, 2H, ArꢀH); 7.20(d, J=7.72 Hz, 1H, ArꢀH);
7.13(t, J=7.24 Hz ,1H, ArꢀH), 6.85(d, J=8.28 Hz, 1H, ArꢀH); 5.76(d, J=4.88 Hz, 1H, ArꢀH);
4.06(t, J=6.46 Hz, 2H, 1×NꢀCH2); 3.72(s,8H, 4×NꢀCH2); 3.49(t, 1H, J=7.84 Hz, 1×NꢀCH2);
2.16~2.24(m, 2H, 1×CH2). HRMS(TOF MS ESI, [Mꢀ2HCl+H]+): m/z calculated for C28H26N3O4
:468.1923, found: 468.1908. Anal.calcd for C28H27Cl2N3O4·3.0H2O: C 56.57%, H 5.60%, N
7.07%; found: C 56.65%, H 5.34%, N 7.03%.
2ꢀ{2ꢀ[(4ꢀpiperazinꢀ1ꢀyl)–aminobutyl]1Hꢀbenz[de]isoquinolineꢀ1,3(2H)ꢀdione}ꢀ6ꢀ4Hꢀ
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chromeneꢀ4ꢀone dihydrochloride (6g). Yield 86%, white solid. H NMR (400 MHz, D2O) δ:
7.60ꢀ7.64(m, 3H, ArꢀH); 7.28(d, J=5.16 Hz, 1H, ArꢀH); 7.17~7.20(m, 2H, ArꢀH); 6.95(s, 2H, Arꢀ
H); 6.62(J=6.32 Hz, d, 1H, ArꢀH), 5.54(s, 1H, ArꢀH); 3.69~3.81(m,10H, 5×NꢀCH2); 3.39(t,
J=8.2 Hz, 2H, 1×NꢀCH2); 1.83ꢀ1.90(m, 2H, 1×CH2); 1.63 (s, 2H, 1×CH2). HRMS(TOF MS ESI,
[M–2HCl+H]+): m/z calculated for C29H28N3O4: 482.2080, found.482.2059. Anal.calcd for
C29H29Cl2N3O4·3.0H2O: C 57.24%, H 5.80%, N 6.91%; found C 57.53%, H 5.64%, N 6.95%.
2ꢀ{2ꢀ[(3ꢀ(morpholinoꢀ1ꢀyl)–propyl]1Hꢀbenz[de]isoquinolineꢀ1,3(2H)ꢀdione}ꢀ6ꢀ4Hꢀchromeneꢀ
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4ꢀone hydrochloride (6h). Yield 86%, white solid. H NMR (400 MHz, D2O) δ: 7.84(d, J=7.56
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Hz ,2H, ArꢀH); 7.77(d, J=7.12 Hz,1H, ArꢀH); 7.48(d, J=7.56 Hz, 1H, ArꢀH); 7.35ꢀ7.39(m, 1H,
ArꢀH); 7.30(t, J=7.82 Hz, 1H, ArꢀH); 7.09ꢀ7.17(m, 2H, ArꢀH); 6.86(d, J=8.28 Hz, 1H, ArꢀH);
5.66(s, 1H, ArꢀH); 4.18(d, J=10.92 Hz, 2H, 1×OꢀCH2); 4.03(t, 2H, J=6.66 Hz, 1×OꢀCH2); 3.89(t,
J=11.78 Hz, 2H, 1×NꢀCH2); 3.61~3.66(m, 2H, 1×NꢀCH2); 3.37(t, J=7.94 Hz, 2H, 1×NꢀCH2);
3.22ꢀ3.28(m, 2H, 1×NꢀCH2); 2.12ꢀ2.20(m, 2H, 1×CH2). HRMS (TOF MS ESI, [MꢀHCl+H]+):
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m/z calculated for C28H25N2O5
:
469.1763, found 469.1748. Anal.calcd for
C28H25ClN2O5·1.85H2O: C 62.48%, H 5.37%, N 5.20%; found C 62.41%, H 5.29%, N 5.17%.
4.2. Biological Materials and Methods. 4.2.1. Cell Lines and Cell Culture. Hepatocellular
cancer cells (HepG2, SMMCꢀ7721) and the normal liver cells (QSGꢀ7701) were cultured in
RPMIꢀ1640 medium supplemented with 10% FBS, penicillin (100 units/ml) and streptomycin
(100 units/ml) in a humidified atmosphere with 5% CO2 at 37°C. These cell lines were
purchased from American type Culture Collection (MD, USA). RPMIꢀ1640 medium and FBS
were obtained from GIBCO (Thermo Fisher Scientific, MA, USA).
4.2.2. Cell Viability Assay. The cytotoxicity of all the compounds in the presence or absence of
spermidine (50 ꢂM) was detected by MTT assays. In brief, cells were seeded into 96ꢀwell plates
at 5×103cells/well. After 12 h, various concentrations (1, 5, 10, 30 and 50ꢂM) of compounds
were subsequently added and incubated for 48 h. Then, cells were incubated with MTT for 4 h at
37°C in a humidified atmosphere with 5% CO2 and were subsequently lysed in dimethyl
sulphoxide. Cells were then vibrated gently for 10 min. The formation of formazan was
measured by using a microtitre plate reader at 590 nm. Growth inhibition by a compound was
evaluated by IC50 (concentration of a drug causing 50% inhibition of cell growth). Each viability
inhibition experiment was repeated at least three times. The concentration of spermidine (50 ꢂM)
was selected for no obvious cytotoxicity on tested cells.
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4.2.3. Intracellular Fluorescence Intensity Assay. The intracellular fluorescence intensity of 6c
was detected by using high content screening (HCS) analysis (Thermo Scientific Cellomics
ArrayScan VTI, Cellomics, Pittsburgh, PA, USA). Briefly, after cells were seeded into 96ꢀwell
plates at 5×103 cells/well for 24 h, 6c (0, 1, 5, 10 and 20 ꢂM) was added into 96ꢀwell plates.
After incubation for 8 h, the cells were stained with acridine orange (AO) for 15 min. Then, cells
were washed three times with PBS. Cell numbers were determined by AO staining and
intracellular fluorescence intensity of 6c was detected. For the measurement of timeꢀdependent
intracellular fluorescence of 6c, cells were seeded into 96ꢀwell plates at 1×104 cells/well. After
incubation for 24 h, 6c (10 ꢂM) was added into 96ꢀwell plates for 0.5, 1, 2, 4, 8 and 12 h. Cells
were washed three times with RPMIꢀ1640 medium, and the images were captured using a Leika
inverted microscope.
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4.2.4. Immunofluorescence Staining. Cells were seeded into 96ꢀwell plates at 1×104 cells/well.
After incubation for 24 h, 6c (1, 5, 10 and 20 ꢂM) was added into 96ꢀwell plates for 24 h. The
cells were fixed using 4% formaldehyde for 30 min. The fixed cells were washed three times
with phosphate buffer saline (PBS) and were blocked in 0.5% bovine serum albumin. The
primary specific antibody of p53 and cleaved caspase 3 was added to the wells. The cells and
antibody were incubated at 4°C overnight, followed by incubation with the secondary antibody.
Then, Hoechst3342 was added to samples, followed by incubation in the dark. Images were
recorded by HCS and the fluorescence intensity was recorded.
4.2.5. Annexin V-FITC/Propidium Iodide Staining. Cell apoptosis was evaluated using Annexin
VꢀFITC/Propidium iodide (PI) staining detected by flow cytometry. Cells were seeded into 6ꢀ
well plates at 1×105 cells/well. After incubation for 24 h, cells were treated with 6c (20 ꢂM) for
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24 h. Then the cells were harvested and stained according to the manufacturer’s protocol.
Analysis was performed by flow cytometry (BD Biosciences, San Jose, CA, USA).
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4.2.6. Reactive Oxygen Species Assay. Reactive oxygen species (ROS) were examined with
fluorescent probe DCFHꢀDA detected by HCS. Cells were seeded into 96ꢀwell plates at 1×104
cells/well and were cultured at 37°C in a humidified atmosphere of 5% CO2 for 24 h. 6c (20 ꢂM)
was added into 96ꢀwell plates. After incubation for 8 h, cells were washed three times with PBS
followed by staining with 10 ꢂM DCFHꢀDA in the dark for 15 min at room temperature. Then
cells were washed three times with PBS, stained with Hoechst33342 and analyzed by HCS. For
the ROS inhibitor analysis, cells were respectively pretreated with 10 mM NAC, 5 mM TOC and
50 µM CAT for 1 h, and measured following the procedure mentioned above.
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4.2.7. Colony Formation Assay. The ability of isolated single cells to form colonies was tested
using two dimensional (2D) culture. Cells were harvested and resuspended using the medium.
Two hundred cells in 100 ꢂL medium were seeded into the individual well of 96ꢀwell plates.
After incubation with 6c for 10 days, the colonies were washed three times with PBS and fixed
with 4 % paraformaldehyde for 20 min. All cells were stained with crystal violet. Experiments
were set up in triplicate and the medium was changed every two days.
4.2.8. Polyamine Level Assay. Polyamine contents of HepG2 and SMMCꢀ7721 cells treated with
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or without 6c were quantified by using HPLC according to published methods (Agilent 1200,
Agilent Technologies, Santa Clara, CA,USA) coupled with a G1321A fluorescence detector. The
polyamines (putrescine, spermidine, and spermine) were converted to the corresponding dansyl
derivatives and separated on a C18 analytical column by eluting with an ethyl acetate/water
gradient. 1,6ꢀDiaminohexane was used as the internal standard. Polyamine levels are expressed
as mg/g protein.
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4.2.9. Western Blot. Western blot assays were performed to detect the expression of related
proteins. In brief, cells were treated with 6c at the concentration of 5, 10 and 20 ꢂM for 24 h, and
were harvested and centrifuged. Cell pellets were washed three times with iceꢀcold PBS and
were lysed with RIPA buffer (Beyotime, Jiangsu, China). The concentration of protein was
examined using BCA assay kit. The total lysates were subjected to 5×SDSꢀloading buffer at
100°C for 10 min followed by exposure to 12% SDSꢀPAGE for 2 h. Extracted proteins were
transferred onto PVDF membranes, which were blocked by 5% dried skimmed milk at room
temperature for 1 h. After blockage, the membranes were incubated with specific primary
antibodies at 4°C overnight. After incubation with determined HRPꢀconjugated secondary
antibody, the protein expression was detected by using the ECL plus reagents (Beyotime, Jiangsu,
China).
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4.2.10. Evaluation of Mitochondria Membrane Potential. Mitochondria membrane potential was
analyzed by staining with JCꢀ1 dye or Rh123 dye. In brief, cells were seeded in 6ꢀwell plates at a
density of 1 × 105 cells/mL and allowed to adhere overnight. Culture medium was replaced with
fresh medium containing 6c (20 ꢂM). After incubation for 24 h, cells were trypsinized, washed
three times with PBS, and incubated with JCꢀ1 dye or Rh123 which were purchased from
Invitrogen (Thermo Fisher Scientific, MA, USA) at a final concentration of 10 ꢂg/mL for
30 min. Then, cells were washed with PBS, centrifuged to remove the supernatant. This
procedure was repeated three times, and the cell pellets were resuspended with fresh medium for
flow cytometric analysis. JCꢀ1 accumulates within intact mitochondria to form multimer Jꢀ
aggregates (red colour) and the red colour of the dye reduced because of depolarization of
mitochondrial membrane potential. For Rh123, the green fluorescence also accumulated in intact
mitochondria. This alteration was analyzed by flow cytometry.
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4.2.11. Examination of Superoxide. Superoxide production was examined using a superoxide
assay kit (Beyotime, Jiangshu, China). Briefly, cells were seeded in 96ꢀwell plates at a density of
1 × 104 cells/well and allowed to adhere overnight. Culture medium was replaced with fresh
medium containing 6c at 20 ꢂM. After incubation for 24 h, cells were washed three times with
PBS. 200 ꢂL of superoxide detection buffer was added to every well, and incubated at 37°C for 3
min. Absorbance was read at 450 nm using the microplate reader.
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4.2.12. Transwell Migration Assay. Transwell migration assays were performed by using
modified Boyden’s chamber in 24ꢀwell cell culture plate with 8ꢀꢂm pore. Chambers were
washed with PBS for three times. Then the medium with the tested compound was placed in the
lower chamber, and cells were seeded in the top chamber. Cells were treated with 6c at 37°C in a
humidified atmosphere of 5% CO2. After incubation for 24 h, nonꢀmigrated cells on the top
surface of the membrane were gently scraped away with cotton swab. The migrated cells was
fixed with 4 % paraformaldehyde for 20 min and stained with 0.2% crystal violet. Images were
recorded using a Leika inverted microscope.
4.2.13. Examination of H2O2 Level. H2O2 level was measured by a hydrogen peroxide assay kit
(Beyotime, Jiangshu, China), following the manufacturer’s instructions. Briefly, 50 ꢂL protein
samples were incubated with the reagent at room temperature for 30 min, and the absorbance at
560 nm was measured. The H2O2 concentration was calculated from a standard curve.
4.2.14. Isolation of Mitochondria. The followed protocol is an adaptation of that used previously.
Approximately 30 million HepG2 cells exposed to 6c (10 ꢂM) for 8 h were harvested from
adherent culture by trypsinization, washed with cold PBS, and resuspended in 1 mL of
mitochondrial extraction buffer (68 mM sucrose, 200 mM mannitol, 50 mM KCl, 50 mM pipes,
5 mM EGTA, 1 mM DTT, 2 mM MgCl2, protease inhibitors). The cell suspension was incubated
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on ice for 15 min and then transfered to a homogenizer by using a 21 gauge needle and 1ꢀmL
syringe. Cells were homogenized for ten times, and trypan blue was used to define the
homogenized effect. At 4°C, the resulting lysate was centrifuged at 1000 g for 10 min. The
supernatant was then transferred to a new microcentrifuge tube and centrifuged at 4°C for an
additional 10 min at 11,000 g. The supernatant was removed and the pellet was saved as the
mitochondrial fraction.
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4.2.15. Detection of Total Glutathione. Total glutathione (containing reduced and oxidized
glutathione) level was measured using GSH and GSSG assay kit according to the manufacturer's
instructions (Beyotime, Jiangshu, China). Briefly, after treatment with 6c (20 ꢂM) for 24 h, cells
were washed twice with PBS by centrifugation at 600 g for 5 min. Pellets were resuspended in
protein removal buffer and vortexed immediately followed by being lysed by three freezeꢀthaw
cycles. After cell lysates were centrifuged at 12,000 g for 10 min at 4°C, supernatant containing
total glutathione was transferred to another tube. For quantifying total glutathione level, 10 ꢂL
supernatant was mixed with 150 ꢂL total glutathione assay buffer for 5 min, and then 50 ꢂL
NADPH (0.16 mg/ml) was added. Absorbance was read at 405 nm by using the microplate
reader.
4.2.16. AO/EB Staining. Treatment of cells with 6c at the concentration of 1, 5, 10, 20 ꢂM for 24
h, cells were washed three times and resuspended in PBS. Cell suspension (200 ꢂL) was mixed
with 8 ꢂL dye mixture containing the same amount of AO (1 mg/mL) and EB (1 mg/mL) (final
concentration of dye mixture: 4 ꢂg/mL). After incubation for 20 min, fluorescence intensity of
AO and EB was detected with HCS. The images of each concentration were captured.
4.2.17. Primary Tumor Growth and Lung Metastasis. All animal care and experimentation
conformed to the Guide for the Care and Use of Laboratory Animals published by Henan
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University. For solid tumor development, male Kunming mice (Laboratory Animal Center of
Henan, Zhengzhou) aged 5 weeks (weighing 18−22 g) were injected subcutaneously with 2 × 106
H22 cells. On the eighth day after inoculation, the mice were randomly divided into three groups
(negative control, 6c group, and amonafide group). Then, the mice were administered by 6c (3
mg/kg) or amonafide (5 mg/kg, as positive control) or physiologic saline (negative control) for 7
consecutive days via tail vein injection. Measurements of body weight were performed daily.
The mice were finally killed by ether anesthesia, and solid tumors were removed and weighed.
The inhibitory rate was calculated as follows: inhibitory rate (%) = [(A − B)/A]×100, where A is
the mean tumor weight of the negative control group, and B is the mean tumor weight of the
drug treated or positive group. Meanwhile, heart, liver, kidney, lung and spleen of the mice were
removed and weighed at the last day. The organ weight index and histopathology were
investigated for systemic toxicity evaluation: organ index (%) = (organ weight/body weight) ×
100.
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H22 cells (3×106 cells per mouse) for Kunming mice were injected through the tail vein for
tumor lung metastasis. To ensure all mice bore actively growing lung tumors before the drug
treatment, pulmonary metastasis was allowed to develop for 7 days. On day 8, mice were
injected by tail vein with 6c (3 mg/kg), amonafide (5 mg/kg, positive control), and normal saline
(negative control) for 7 consecutive days. On day 15, mice were killed by ether anesthesia and
lungs were removed. After fixed by 4% paraformaldehyde for one day, lung metastases nodules
was counted. Fixed tissues by 4% paraformaldehyde were sectioned at 5 ꢂm. Hematoxylin and
eosin (H&E) staining was performed according to a standard protocol. Images were taken using
a Leica microscope (Leica, Wetzlar,Germany).
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4.2.18. Data Analysis. All the data are presented as the mean±SD and analyzed using Student’s tꢀ
test or analysis of variance (ANOVA) followed by qꢀtest. (*) p< 0.05, (**) p< 0.01 and (***) p<
0.001 were considered to be significant statistically.
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AUTHOR INFORMATION
Corresponding Author
* For Y.W.: phone, 86ꢀ18739998722; eꢀmail: wangyuxia@henu.edu.cn. For S.X.: phone, 86ꢀ
13938637212; eꢀmail: xiesq@henu.edu.cn. For C.W.: phone, 86ꢀ13619810550; fax, 86ꢀ371ꢀ
22864665; eꢀmail: wcjsxq@henu.edu.cn.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by National Science Foundation of China (21172053, 81573465),
Program for Science and Technology Innovation Talents in Universities of Henan Province
(14HASTIT033), Projects of Science and Technology of Henan (13420051009, 162300410231,
152300410058), China Postdoctoral Science Foundation Funded Project (2015M582183),
Postdoctoral Research Sponsorship of Henan Province (2015035).
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ABBREVIATIONS
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ROS, reactive oxygen species; HCS, high content screening; PAT, polyamine transporter; H&E,
hematoxylin and eosin; AO, acridine orange; EB, ethidium bromide; DCFHꢀDA, 2',7'ꢀ
dichlorodihydrofluorescein diacetate (DCFHꢀDA); MMP, mitochondrion membrane potential;
MTT, 3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide; TOC, alphaꢀ tocopherol;
CAT, catechin; H&E, hematoxylin and eosin; PI, propidium iodide; HCC, hepatocellular
carcinoma.
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