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Introduction
The naturally occurring polyamines, namely putrescine, spermidine, and spermine, play a crucial role in cell
growth[1], such as the activation of kinases involved in the signal transduction pathway, the regulation of ion channel gating, and the
modulation of oxidative processes[2]. Cells have complex regulatory mechanisms that control intracellular polyamine levels
via transport, degradation, and
biosynthesis[3]. Rapidly-dividing cells need large amounts of polyamines in
order to grow, which can be realized by internal biosynthesis and/or external uptake via the active polyamine transporter
(PAT). The elevated requirement of tumor cells for polyamines makes the polyamine pathway attractive for
tumor-targeted chemotherapy[4].
Polyamines are also potential carriers for drug delivery
because the polyamine transporter in tumor cells seems to
have a wide structural tolerance of the substrates. In this
regard, the antineoplastic drugs are conjugated to polyamines
to facilitate their entrance into cells with active PAT. Several
related polyamine-drug conjugates were designed to increase
the cytotoxicity to tumor cells and reduce the side-effects on
normal cells[5]. Previous efforts revealed that the non-native
triamine, homospermidine, showed a better PAT recognition
than natural polyamines. A leading conjugate,
anthracenyl-methyl homospermidine (ANTMHspd), was found to
display an excellent PAT selectivity and strong inhibitory
effects on several tumor cell lines[6]. Many reports revealed
that alkyl polyamine analogues exhibited cytotoxicity via
apoptosis[7,8], however, the antiproliferative mechanism of
the homospermidine conjugates has not been elucidated until
now. In this study, the antitumor potency of ANTMHspd
was investigated in the human hepatoma BEL-7402 cell line.
The data demonstrated that ANTMHspd inhibited growth
effectively and induced apoptosis in BEL-7402 cells.
Materials and methods
Materials All chemicals were purchased from Sigma (St
Louis, MO, USA), unless otherwise indicated. RPMI-1640
and fetal calf serum (FCS) were purchased from Gibco (Grand
island, NY, USA). The primary antibodies against
caspase-3, caspase-8, caspase-9, cytochrome c, Bcl-2, Bax, as well as
peroxidase-conjugated goat antimouse or antirabbit
secondary antibody, were purchased from Santa Cruz
Biotechnology (Delaware Avenue Santa Cruz, CA, USA). Z-DEVD-fmk
(caspase-3 inhibitor) and Z-LEHD-fmk (caspase-9 inhibitor)
were purchased from Imgenex (Suite E San diego, CA, USA).
ANTMHspd (Figure 1) was provided by Prof WANG (Institute of Natural Products and Medicinal Chemistry,
Kaifeng, China). The stock solution (10 mmol/L) was
prepared in DMSO and diluted to various concentrations with
serum-free culture medium.
Cell culture The human hepatoma BEL-7402 cell line,
obtained from American Type Culture Collection (ATCC,
Rockville, MD, USA), was cultured in RPMI-1640
supplemented with 10% heat-inactivated FCS and antibiotics (100
units/mL penicillin and 100 µg/mL streptomycin sulfate) at
37 °C in an atmosphere of 95% air and 5%
CO2 under humidified conditions. 1 mmol/L aminoguanidine was added as an
inhibitor of amine oxidase derived from FCS and had no
effect on the various parameters of the cells measured in this
study[9].
Evaluation of cell viability and proliferation
MTT assay Chemosensitivity was assessed using the
thiazolyl blue tetrazolium bromide (MTT)
assay[1]. Briefly, 5000 exponentially growing cells were seeded onto 96-well,
flat-bottomed plates and allowed to attach overnight. The
cells were treated with the indicated concentrations of
ANTMHspd for 48 h and 100 µL MTT (1 mg/mL) was added
to each well. After incubation at 37 °C for 4 h, the MTT
solution was removed and the crystals of the viable cells
were dissolved with DMSO. The absorbance of each well
was read at 570 nm.
Growth inhibition assay The exponentially growing cells
were seeded onto 24-well, flat-bottomed plates at a density
of 5×104/mL and allowed to attach overnight. The cells were
treated with the indicated concentrations of ANTMHspd for
different times. The cells were collected by trypsinization at
different time points (d 1, 2, 3, 4, and 5) and counted by a
Thoma hemocytometer (Shanghai, China) using the trypan
blue dye exclusion method for viability.
Apoptosis and cell cycle analysis Apoptosis
was quantified by assessing the fraction of cells with a
sub-G1 DNA content by flow cytometry. The cells were seeded in
25cm2 flasks and then pre-incubated in RPM-I1640 supplemented
with 0.2% FCS for 24 h which induced cell cycle
synchronization[10]. The synchronous cells were treated with the
indicated concentrations of ANTMHspd. After incubation for
24 or 36 h, the cells were washed twice with ice-cold
phosphate-buffered solution (PBS), fixed, and permeabilized with
ice-cold 70% ethanol at -20 °C overnight. The cells were
treated with 50 µg/mL RNase A at room temperature for 30
min after being washed with ice-cold PBS, and finally stained
with 50 µg/mL propidium iodide (PI) in the dark at 4 °C for 30
min. The distribution of the cell cycle phases with different
DNA contents was read in a flow cytometer. Ten thousand
events were acquired in each sample[11].
Hoechst staining of nuclear chromatin The cells were
fixed with 4% formaldehyde in PBS at 37 °C for 10 min and
permeabilized with a 19:1 mixture of ethanol/acetic acid at
-20 °C for 15 min. The fixed cells were stained with 1
µg/mL Hoechst 33258 in PBS at room temperature for 20 min. The
Hoechst-stained cells were analyzed by fluorescence
microscopy[1].
Assessment of the change in mitochondrial membrane
potential The mitochondria membrane potential (MMP) was measured using flow cytometry with rhodamine
123 (Rh123) and PI double staining. Rh123 accumulates in
normal mitochondria due to its high negative charge and the
reduction of MMP leads to the release of Rh123. Rh123 was
dissolved in DMSO and diluted in PBS before treatment.
About 1×106 cells were harvested by trypsinization, washed
twice with PBS, and incubated with Rh123 (10 µg/mL) at 37
°C for 30 min in the dark. PI (10 µg/mL) was then added to the
cells and the cells were incubated for 5 min in the dark. The
samples were analyzed by flow cytometry with a 15 mw
argon laser at 488 nm. Fluorescence intensity was detected
at the wavelength of 575 nm and 610 nm, respectively. The
percentage of Rh123_/PI+ and
Rh123_/PI_ presented the
effective collapsed MMP[12].
Caspase activity assay The cell pellets were washed
with ice-cold PBS and resuspended in 100 mmol/L Hepes
buffer (pH 7.4), which contained protease inhibitors (5
µg/mL aprotinin and pepstatin, 10 µg/mL leupeptin, and 0.5
mmol/L phenylmethanesulfonyl fluoride). The cell
suspension was lysed by 3 freeze-thawed cycles and the cytosolic
fraction was obtained by centrifugation at 12
000×g at 4 °C for 20 min. DEVDase, IETDase, and LEHDase activities were
evaluated by measuring proteolytic cleavage of
chromogenic substrates Ac-DEVD-pNA, Ac-IETD-pNA, and
Ac-LEHDpNA, which were used as the substrates for caspase-3, caspase-8, and caspase-9-like proteases,
respec-tively. Briefly, the cell lysate (50 mg of protein) was added
into the buffer containing 150 mol/L Ac-DEVD-pNA,
Ac-IETD-pNA, and Ac-LEHD-pNA to a final volume of 150 µL.
The reaction mixture was incubated at 37 °C for 1 h. The
absorbance of enzymatically-released pNA was measured at
405 nm on a microplate reader every 20
min[13].
Western blotting The cells, treated with different
concentrations of ANTMHspd for the desired exposure time,
were harvested by trypsinization and washed with PBS. For
the caspase inhibitor analysis, the cells were pretreated with
50 µmol/L Z-DEVD-fmk or Z-LEHD-fmk for 2 h, and then
exposed with ANTMHspd for 24 h. Cytosolic and
mitochondrial fractions were prepared as
described[14]. Briefly, the cells were resuspended in 300 µL buffer (2 mmol/L
Hepes-potassium hydroxide (KOH) [pH 7.5], 10 mmol/L
MgCl2, 1 mmol/L ethyleneglycol bis(2-aminoethyl ether)tetraacetic
acid (EGTA), 1 mmol/L dithiothreito (DTT), and 250 mmol/L
sucrose and protease inhibitor). After homogenization, the
unbroken cells, large plasma membrane pieces, and nuclei
were removed by centrifugation at
1000×g for 10 min. The supernatant was subjected to centrifugation at
10 000×g for 20 min. The pellet fraction containing mitochondria was
resuspended in 500 µL buffer (10 mmol/L Tris-acetate [pH 8.0],
0.5% Nonidet NP-40, and 5 mmol/L CaCl2). The supernatant
was further centrifuged at 50 000×g for 2 h to generate cytosol.
The detection of cytochrome c in the cytosolic and
mitochondria fractions was analyzed by Western blotting. Total
cellular protein was isolated using the protein extraction
buffer (containing 150 mmol/L NaCl, 10 mmol/L Tris [pH 7.2],
5 mmol/L EDTA, 0.1% Triton-100, 5% glycerol, and 2% SDS).
The protein concentrations were determined using the
protein assay kit. Equal amounts of proteins (50 µg/lane) were
fractionated using 12% SDS-PAGE and transferred to
polyvinylidene difluoride (PVDF) membranes. The
membranes were incubated with primary antibodies against
caspase-3, caspase-8, caspase-9, cytochrome c, Bcl-2, and
Bax (1:5000). After being washed with PBS, the membranes
were incubated with peroxidase-conjugated goat antimouse
or antirabbit secondary antibody (1:3000), followed by
enhanced chemiluminescence staining through the enhanced
chemiluminescence system. Actin was used to normalize
protein loading[1].
Statistical analysis Results from at least 3 independent
experiments were given as mean±SD. Statistical significance
was done using the ANOVA test. Results were considered
significant when P<0.05.
Results
Inhibition of growth by ANTMHspd In the present study,
ANTMHspd exhibited a dose- and time-dependent growth
inhibition against BEL-7402 cells at the dose of 0.1_50
µmol/L (Figure 2). The 50% inhibiting concentration
(IC50) value for the BEL-7402 cells was 0.56±0.08 µmol/L for a
48 h treatment. The proliferation of the BEL-7402 cells was gradually
attenuated with the prolonged treatment time of ANTMHspd
(Figure 3).
Cell cycle perturbation by ANTMHspd A flow cytometry
DNA analysis revealed that ANTMHspd induced cell cycle
perturbation (Figure 4). The detailed data concerning the
percentage of sub-G1 cells and cell cycle distribution of
BEL-7402 cells treated with ANTMHspd are shown in Table 1.
Compared to the control (untreated cells), changes in the
cell cycle distribution of treated BEL-7402 cells were evident.
This was accompanied by an increase in the
sub-G1 region of cells with a fractional DNA content.
Effect of ANTMHspd on cell morphology To determine
whether or not the sub-G1 fraction, affected by ANTMHspd,
was related to apoptosis, both the control and
ANTMHspd-treated cells were stained with the fluorescent dye Hoechst
33258 and visualized by fluorescence microscopy. Typical
morphological changes of apoptosis, including chromatin
condensation, dense chartreuse nucleolus, and nuclear
fragmentation, were observed in the treated cells, but not in
the control cells (Figure 5).
Assessment of the change in MMP In Figure 6, the lower
left quadrant represented the percentage of
Rh123_/PI_ cells,
the higher left quadrant represented the percentage of
Rh123_/PI+ cells, the lower right quadrant represented the
percentage of
Rh123+/PI_ cells, and the higher right quadrant
represented the percentage of
Rh123+/PI+ cells which reflected
the damaged cells. Most untreated cells have intact plasma
membrane and normal MMP. After being treated with the
indicated concentrations of ANTMHspd for 12 or 24 h,
Rh123_/PI_ and
Rh123_/PI+ cells exhibited a dose- and
time-dependent increase (Figure 6). These results indicated that
BEL-7402 cells lost MMP and membrane integrity. For
example, the percentage of MMP collapse reached 70.25%
after the cells were treated with 5 μmol/L ANTMHspd for
24 h (Table 2).
Effect of ANTMHspd on apoptosis-related
proteins The activity of caspase-9 and caspase-3 was significantly
increased compared with untreated cells in a dose- and
time-dependent manner, while ANTMHspd had no effect on the
activity of caspase-8 (Figure 7). These results were
confirmed by Western blotting. ANTMHspd treatment increased
the cleavage of caspase-9 and caspase-3 in a dose- and
time-dependent manner. The expression of cleaved-caspase-8
was undetectable in ANTMHspd-treated cell and was
detected in the positive control cell
(5×108 U/L TNF-α-treated cell). The cytochrome c release from mitochondria was
enhanced concomitant with the related attenuation of
cytochrome c in mitochondria. The Bcl-2 protein expression was
downregulated and Bax was upregulated from 24 to 48 h
(Figure 8). In addition, the specific inhibitors of caspase-9
and caspase-3 almost completely abolished the
ANTMHspd-induced cleavage of caspase-9 and caspase-3, respectively
(Figure 9).
Discussion
A recent review pointed out new polyamine derivatives
as potent therapeutic agents[15]. The roles of alkyl polyamine
analogues in apoptosis have been investigated in a number
of experimental systems. The results indicated that polyamine
analogues could produce cytotoxicity through
apoptosis[7,8], but the related functions of polyamine conjugates are rarely
elucidated.
Some data demonstrated that polyamine analogues
activated a classical apoptosis response, including the release
of cytochrome c and the activation of the caspase cascade,
which initiated the morphological and biochemical steps in
the apoptosis pathway[16]. In this study, we found that
ANTMHspd could inhibit BEL-7402 cell proliferation with
an IC50 value of 0.56±0.08 µmol/L at 48 h. This revealed that
ANTMHspd might be a potential leading agent with a
structure different from the alkyl polyamine
analogues[1]. To explore the mechanism responsible for the antiproliferative
effects of ANTMHspd, the changes of cell morphology were
first assessed. After being treated with different
concentrations of ANTMHspd, the morphology of BEL-7402 cells
changed obviously, including cell shrinkage, nuclear
fragmentation, as well as chromatin condensation. A flow
cytometry DNA analysis revealed that ANTMHspd induced
a sub-G1 cell population and cell cycle perturbations. These
results indicated that ANTMHspd could induce BEL-7402
cell apoptosis.
Apoptosis can be triggered by several stimuli and is
controlled by 2 major pathways, namely the mitochondrial
pathway and membrane death receptor
pathway[17]. In the mitochondrial pathway, mitochondria have a crucial position in
apoptosis control. The loss of MMP induces cytochrome c
release from the mitochondria to the cytoplasm, which leads
to the activation of caspase-9 and downstream cleavage of
caspase-3. The membrane death receptor pathway is
characterized by the binding between cell death ligands and cell
death receptors and the subsequent activation of caspase-8
and caspase-3.
To reveal the precise molecular mechanism of
ANTMHspd-induced apoptosis in BEL-7402 cells, we observed the effect
of ANTMHspd on MMP, cytochrome c, the activity of caspases, as well as the Bcl-2 family. The present results
showed that ANTMHspd decreased MMP and increased the level of cytochrome c in the cytoplasm with the
corresponding decrease of cytochrome c in mito-chondria. To
determine whether caspases are involved in the
ANTMHspd-induced apoptosis in BEL-7402 cells, the catalytic activity of
caspase-8, caspase-9, caspase-3, as well as their expression
were measured. The data demonstrated that ANTMHspd
could activate caspase-9 and caspase-3. However, the
activity of caspase-8, an apoptosis-initiating protease linked
to the death receptors, was unchanged and the cleaved
fragment of caspase-8 was not detected. It was suggested that
ANTMHspd induced apoptosis via the mitochondrial pathway, but not the membrane death receptor pathway.
Furthermore, to determine whether the activation of caspases
is essential for ANTMHspd-mediated apoptosis,
pre-incubation with specific inhibitors of caspase-9 and caspase-3
almost abolished the ANTMHspd-induced caspase-9 and
caspase-3 activation respectively. This confirmed that
ANTMHspd induced apoptosis in a caspase-dependent
manner. However, BE-3-3-3, a bis(ethyl)norsper-mine,
induces breast cancer cell apoptosis via both the membrane
death receptor pathway and mitochondrial pathway because
BE-3-3-3 can activate caspase-9, caspase-3, and caspase-8
in human breast cancer cells[18]. This different pathway
demonstrates that the ability of polyamine analogues to induce
apoptosis is dependent on the characteristics of the cell line.
The Bcl-2 family plays a central role in regulating the
mitochondrial apoptosis pathway. More than 20 Bcl-2
family members consist of anti-apoptosis membranes (including
Bcl-2 and Bcl-xL) and pro-apoptosis membranes (including
Bax and Bak) have been identified. Bcl-2 is an important
element during apoptosis mediated by the mitochondrial
pathway and has been identified as preventing cytochrome
c release from the mitochondria. In contrast, Bax can induce
the release of cytochrome c from the
mitochondria[19]. The present report reveals that ANTMHspd-induced apoptosis
is companied by an increased expression of Bax and a
reduced protein level of Bcl-2 in BEL-7402 cells. Huang et al
reported that other pathways but caspases can function as
apoptosis effectors in brease cancer (MDA-MB-231 and
MCF7) cells and that the regulation of Bcl-2 family members
by polyamine analogue was cell type
specific[1]. The effects of ANTMHspd on caspase activity and the expression of
the Bcl-2 family in other cell lines will be carried out in our
laboratory.
Taken together, we can conclude that ANTMHspd
induced the apoptosis of BEL-7402 cells via the
mitochondria/caspase-9/caspase-3 dependent pathway and the Bcl-2
family was involved in the control of apoptosis.
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