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Introduction
Hepatocellular carcinoma (HCC), one of the most
common malignant tumors worldwide, is often diagnosed at an
advanced stage when most potentially curative therapies
such as resection, transplantation, or percutaneous and
transarterial interventions are of limited
efficacy[1_4]. The fact that HCC is resistant to conventional chemotherapy,
and is rarely amenable to radiotherapy, leaves this disease
with no effective therapeutic options and poor
prognosis[5_7]. Therefore, the development of more effective therapeutic
tools and strategies is much needed. Despite the
phenotypically and genetically heterogeneous tumors of HCC,
recent insights into the biology of HCC suggest that certain
signaling pathways and molecular alterations are likely to
play essential roles in HCC development by promoting cell
growth and survival. The identification of such mechanisms
may open new avenues for the prevention and treatment of
HCC through the development of targeted therapies.
Dysregulation of growth factors, receptors and their
downstream signaling pathway components represent a
central pro-tumorigenic principle in human hepatocarcinogenesis.
In particular, the insulin-like growth factor/IGF-1 receptor
(IGF/IGF-1R), hepatocyte growth factor
(HGF)[8], Wnt[9], transforming growth factor alpha/epidermal growth factor
receptor (TGF-α/EGFR)[10], and
TGF-β/TGF-βR[11,12] pathways contribute to proliferation, anti-apoptosis, and invasive
behavior of tumor cells. Midkine (MK) was first identified in
embryonic carcinoma cells in early stages during retinoic
acid-induced differentiation[13]. It has been reported that
MK is overexpressed in HCC[14_16], whereas in normal adult
tissues, MK is low or
undetectable[17,18]. MK promotes the
survival[19,20],
growth[21,22], and
migration[23_25] of many cells, which in combination may contribute to oncogenesis and
tumor progression. Therefore, MK may be a promising
target for cancer therapy. In fact, several studies have been
reported that antisense targeting MK inhibits tumor growth,
such as human prostate cells, colon carcinoma
cells[26], and mouse rectal carcinoma
cells[27].
We previously confirmed that MK-AS could significantly
inhibit growth of hepatocellular cells including HepG2,
SMMC-7721, and BEL-7402[28]. The aim of this study was to
evaluate the in vivo antitumor effects of MK-AS in an
in situ human hepatocellular carcinoma model in mice.
Materials and methods
Antisense oligodeoxynucleotides and drugs Antisense
phosphorothioate oligonucleotide MK-AS
(5'-CCCCGGGC-CGCCCTTCTTCA-3') targeting against 108 to 127 base
position of MK mRNA and MK-Sen phosphorothioate
oligonucleotide (5'-TGAAGAAGGGCGGCCCGGGG-3') were
synthesized by an Applied Biosystems Model 391 DNA
synthesizer on solid supports using Oligo Pilot II DNA (Amersham,
Piscataway, NJ, USA) and purified by HPLC, (Waters Delta
Prep 4000, Milford, MA, USA) with SOURCE 15Q
(Amer-sham). The purity of the oligonucleotides was over 95%.
The sense sequence was used as a control. 5-fluorouracil
(5-Fu), purchased from the Shanghai Donghaipu
Pharmaceuticals Company (Shanghai, China), was used as a
positive control.
Animals and in situ human HCC model
The virgin female Balb/c mice used in this experiments were obtained from
the Academy of Military Medical Science (Beijing, China).
All animal experiments were carried out according to the
standards of animal care as outlined in the NIH guide for the Care
and Use of Laboratory Animals. The human HCC tumor
model was described previously[29]. Briefly, the HCM-Y89
tumor derived from a surgical specimen of HCC was cut into
1 mm×1 mm×1 mm tissues, and implanted into the liver of
mice. Twenty days later, the mice treated with or without
drugs were all killed. The tumors were removed and fixed in
neutral buffered 10% formalin, processed by standard
methods, embedded in paraffin, sectioned and stained with
hematoxylin-eosin (HE). It is important to note that the HCC
model maintains various features similar with clinical liver
cancer patients including local growth, regional invasion,
lymph nodes and pulmonary metastasis, peritoneal seeding
with bloody ascites, and secretion of alpha-fetoprotein (AFP)
in the recipient animals[29].
Treatment of in situ HCC xenograft with MK-AS and
anticancer drugs Two days after the in
situ HCC models were established, the tail veins of the mice were injected with
saline (vehicle control), MK-AS (25, 50, and 100
mg·kg_1·d_1) or (5-Fu10
mg·kg_1·d_1) everyday for 20 d. Each group had 8
mice. The body weight and general physical status of the
animal were recorded daily. At the endpoint of the study,
the mice were killed by cervical dislocation and the tumors
were removed and weighed. The tumor sizes were
monitored with calipers; the tumor volume
(V, mm3) was calculated as
(L×W2)/2, where
L=length (mm) and W=width (mm). The percentage of tumor growth inhibition was calculated
as: Inhibitory rate
(%)=(Wcontrol-Wtreat
)/Wcontrol×100.
Histopathological analysis The tumor tissues were
excised and fixed in 10% buffered formalin. Representative
fragments were embedded in paraffin, and stained with HE
for microscopic observations.
Detection of plasma AFP concentration Animal serum
was collected after the mice were killed. Then, the plasma
AFP concentrations were detected by radioimmunoassay
(RIA) according to the manufacturer's instructions.
Western blot analysis The tumor tissues were lysed
with lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 0.5 mmol/L
EDTA, 0.5% NP40, and 150 mmol/L NaCl] in the presence of
protease inhibitors. The lysates were centrifuged at 15 000
×g for 15 min to remove debris. Proteins sample (30 µg) were
separated by 12% SDS-PAGE gel and transferred onto
polyvinylidene difluoride (PVDF) membranes
(Hybond-polyvinylidene difluoride membranes, Amersham). p53, Bax,
Bcl-2, caspase-3 and the MK protein were identified using
the relative primary antibody (Santa Cruz Biotechnology,
Santa Cruz, CA, USA). The reactive band was visualized
with an ECL-plus Detection Kit (Amersham) and scanned by
Gel Doc 1000 (Bio-Rad, Hercules, CA, USA). β-actin was
used as a control.
Statistical analysis Data were expressed as mean±SD.
Statistical analysis was carried out using
Student's t-test (two-tailed); P<0.05 indicated statistical significance.
Results
Effects of MK-AS treatment on in situ HCC xenograft
growth In the present study, we used an in
situ mouse HCC model to evaluate the antitumor activity of MK-AS. In order
to exclude the nonspecific effect of oligonucleotides, sense
sequence (MK-Sen) was used as a control. After
establishing the model for 2 d, MK-AS with different doses (25, 50,
and 100
mg·kg-1·d-1), MK-Sen with 50
mg·kg-1·d-1, 5-Fu with
10 mg·kg-1·d-1, and saline were administered through the tail
vein for 20 d. The tumors were removed after the mice were
killed. The tumors were then measured and weighed. Table
1 shows the final tumor volumes after a 20 d treatment.
Results showed that the tumor volumes decreased in the
MK-AS treatment group compared with the saline control group
(P<0.01). However, for the sense
oligonucleotide treatment group, there was also a clear decrease in tumor
volume compared with the saline group
(P<0.05). No inflammatory infiltrate was observed surrounding the solid tumor (data not
shown). In addition, MK-AS treatment also resulted in a
significant inhibition of tumor weight compared to the sense
or saline-treated mice (Table 2). The highest inhibitory
efficacy was a 65.89% contrast to the saline group, but for the
sense oligonucleotide, the inhibitory rate was only a 19.38%
contrast to the saline group. The inhibitory rate of 50
mg·kg-1·d-1 was 50.39% compared to the sense treatment group
(P<0.01). The effect of MK-AS on the inhibition of tumor proliferation
was dose-dependent (Table 2). The mean body weight of
the mice was not significantly different between the groups
from the beginning to the end (data not shown). It is
reported that a series of guanosine-rich, phosphodiester
oligodeoxynucleotides can strongly inhibit proliferation in a
number of human tumor cell lines[30]. On the basis, in this
experiment, we thought that the induced decline of tumor
volume and weight by MK-Sen might be related to its
guanosine-rich, which is due to its binding activity with some
nucleolin.
Effect of MK-AS treatment on MK expression in
in situ hepatocellular carcinoma
xenografts Our previous studies showed antisense oligonucleotides targeting against MK
downregulated MK expression in hepatocellular carcinoma
cells. Then, we measured the effect of MK-AS on MK
expression in in situ hepatocellular carcinoma xenografts.
The results indicated that the MK-AS efficiently decreased
MK protein content in a dose-dependent manner (Figure 1).
At the same time, we also observed that 5-Fu showed no
effect on MK expression.
Histopathological analysis The morphology of tumors
from MK-AS, MK-Sen, 5-Fu, and the saline-treated mice were
evaluated by HE staining of the histological sections. The
tumors were excised at the endpoint of treatment of each
protocol. Figure 2 shows the representative sections of
tumors from each experimental group. The tumors from the
mice treated with MK-AS or 5-Fu showed a marked increase
in the necrotic area compared with the saline-treated or
normal animals, which suggested that MK-AS treatment induced
HCC necrosis in vivo.
Inhibition of plasma AFP secretion with MK-AS
treatment AFP is often expressed highly in fetal liver, the
gastrointestinal tract, and the yolk sack, but it is
transcriptionally downregulated after birth, and frequently re-expressed
in HCC. Therefore, it is often used as an indicator of
HCC[29]. In this experiment, we used RIA to detect serum AFP
concentration at the endpoint of treatment. Table 3 shows
that MK-AS significantly decreased AFP secretion at 100,
50, and 25
mg·kg-1·d-1 compared with the saline group,
indicating that there were fewer liver tumor cells in the MK-AS
treated mice and reduced circulating AFP.
Effect of MK-AS on apoptosis-related protein
expression Recently, apoptosis has been implicated as one of the
end points of cells exposed to chemotherapeutic agents. p53,
Bax, and Bcl-2 are involved in chemotherapy-induced
apoptosis, but in a cell type-dependent
manner[31_34]. In this experiment, we also measured the effect of MK-AS on these
proteins' expression. Interestingly, we observed that Bax,
caspase-3, and p53 were upregulated in the MK-AS- and
5-Fu-treated groups, however, Bcl-2 decreased. These data
suggested that modulation of pro-apoptotic and apoptotic
protein expression was an important mechanism for MK-AS
inhibiting tumor growth.
Discussion
In the present study, we first analyzed the effect of
MK-AS administration on MK protein content in an
in situ human hepatocellular carcinoma model. As measured by
Western blotting, the MK-AS compounds efficiently
down-regulated the MK expression level in a dose-dependent
manner. However, MK-Sen and 5-Fu showed no effect on
MK expression (Figure 1). Then, we determined the
systemic administration of an in situ mouse HCC model. The
results indicated that MK-AS administration resulted in a
significant inhibition of the in situ mouse HCC model.
Additionally, the histopathological results indicated that
MK-AS could induce tumor necrosis. It needs to be noted
that the experimental model of HCC has some advantages
compared with the inoculated tumor
model[29]. The tumor line originates from human HCC. It maintains the complete
characteristics of human HCC tissue, such as AFP secretion
and drug sensitivity. In addition, the pathological evidence
also suggests that this model exhibits various features seen
in clinical HCC patients. Therefore, the information provided
by this model can reflect the true clinical results of patients
in some ways. AFP is a secretary protein that is
heterogeneously glycosylated.
AFP is usually expressed at high concentrations as
mentioned earlier. It is transcriptionally downregulated after birth
and frequently re-expressed in HCC. Therefore, it is always
used as a diagnostic marker for tumors. Then we analyzed
the drug treatment of AFP content in the serum. The results
showed that serum AFP secretion was significantly
inhibited in a dose-dependent manner in the MK-AS or 5-Fu
treatment groups compared with the saline group. This result
provided evidence that MK-AS could restrain HCC in
vivo.
Up until now, there have been several reports about the
mechanisms of pro-tumorigenesis by MK. It is reported that
MK activates mitogen-activated protein kinase (MAPK)
pathways and promotes cell growth[35]. Additionally, MK
also activates extracellular signal-regulated kinases 1 and 2,
which are well known as signal transducers acting
downstream several receptors[36]. The activated MAPK pathway
was thought to downregulate caspase-3 activity in
neurons[35]. It was also believed that MK could induce phosphorylation
of protein kinase B (AKT, Ser473 and Thr308), which
promotes a series of anti-apoptosis pathways in cells.
Interest-ingly, we also observed that MK-AS modulated expression
of several proteins including Bax, Bcl-2, p53, and caspase-3
in vivo (Figure 3). We observed that caspase-3 protein
content increased in MK-AS treated groups (Figure 3). The p53
tumor-suppressor gene is involved in cell growth control,
arrest, and apoptosis. It was reported that, whatever p53
status, 5-Fu altered p53 transcriptional and translational
regulation leading to the upregulation of the p53 protein.
Moreover, after 5-Fu exposure, Bax and Bcl-2 protein
regulation was under p53 protein control, and Bcl-2 or Bax
induction and the Bcl-2/Bax protein ratio correlated to 5-Fu
sensitivity[34]. In our experiments, we also observed these similar
results. Furthermore, we observed that MK-AS
administration increased p53, Bax, and caspase-3 protein content and
decreased Bcl-2 content. This suggest that MK-AS has some
similar functions with 5-Fu to restrain HCC by inducing some
pro-apoptosis-related protein expression and inhibiting
anti-apoptotic protein expression. It is well accepted that there is
a significant correlation between chemosensitivity and
Bcl-2 to the Bax ratio[34]. However, MK-AS administration can
decrease the ratio in our experiment, so MK-AS can be used
in combination therapy on human tumors.
In summary, we have for the first time directly addressed
the potential therapeutic role of MK-AS in an in
situ human HCC model. Significant inhibition of HCC growth is achieved
by MK-AS, indicating that MK-AS has the possibility to be
developed as an effective antitumor agent.
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