Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 819-829
doi:10.1111/j.1745-7270.2008.00456.x
Down-regulation of Sonic hedgehog signaling
pathway activity is involved in 5-fluorouracil-induced apoptosis and motility
inhibition in Hep3B cells
Qiyu Wang1#, Shuhong Huang2#, Ling Yang1, Ling Zhao2, Yuxia Yin2, Zhongzhen Liu1, Zheyu Chen2*, and Hongwei Zhang1*
1
School of Life Sciences,
Shandong University, Jinan 250100, China
2
School of Medicine,
Shandong University, Jinan 250012, China
Received: March 25,
2008
Accepted: June 26,
2008
This work was supported by the grants from the National Natural
Science Foundation of China (30570967, 30570967, 30228031, 30671072, 30671050
and 30725020) and the National Ministry of Science and Technology of China
(2006CB503803, 2007CB947100 and 2007CB815800)#
These
authors contributed equally to this work
*Corresponding
authors:
Hongwei Zhang: Tel,
86-531-88364935; Fax, 86-531-88565610; E-mail, [email protected]
Zheyu Chen: Tel, 86-531-88382329;
Fax, 86-531-88382329; E-mail, [email protected]
The Sonic hedgehog (SHh) pathway plays a
critical role in normal embryogenesis and carcinogenesis, but its function in cancer
cells treated with 5-fluorouracil (5-FU) remains unknown. We examined the
expression of a subset of SHh signaling pathway genes, including SHh, SMO,
PTC1, Su(Fu) and HIP in human hepatocellular carcinoma
(HCC) cell lines, Hep3B and HepG2, treated with 5-FU by reverse transcription-polymerase
chain reaction. Using trypan blue analysis,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and terminal
deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay,
we also detected the apoptosis of Hep3B cells resulting from the transfection
of pCS2-Gli1 expression vector combined with 5-FU treatment. The motility of
the cells was detected by scratch wound closure assay. The expression and
subcellular location of PTC1 protein in Hep3B cells treated by 5-FU were also
investigated by Western blot analysis and immunofluorescent microscopy. The
results indicated that the expression of SHh pathway target molecules at both
messenger RNA and protein levels are evidently down-regulated in Hep3B cells
treated with 5-FU. The overexpression of Gli1 restores cell viability and, to
some extent, the migration abilities inhibited by 5-FU. Furthermore, 5-FU
treatment affects the subcellular localization of PTC1 protein, a key member
in SHh signaling pathway. Our data showed that the down-regulation of SHh
signaling pathway activity was involved in 5-FU-induced apoptosis and the
inhibition of motility in hedgehog-activated HCC cell lines. This implies that
the combination of SHh signaling pathway inhibitor and 5-FU-based chemotherapy
might represent a more promising strategy against HCC.
Keywords Sonic hedgehog signaling pathway; hepatocellular
carcinoma; 5-fluorouracil; cell apoptosis; cell motility
Hepatocellular carcinoma (HCC), the major tumor type of liver
cancer, is a malignancy with worldwide significance [1,2]. In recent years?liver cancer had the second
highest mortality rate of all malignancies in China, which is approximately 20–40/100,000 per
year. Because the majority of patients present advanced or unresectable HCC,
traditional chemotherapy is ineffective. Therefore, it is necessary to
investigate the molecular mechanism of HCC development in order to create new
approaches to effective therapy [1].The hedgehog (Hh) signaling pathway plays a critical role in
organizing cell growth and differentiation during embryonic tissue patterning
[3]. The role of the Hh pathway in human cancers has been established [4–11]. A variety
of human cancers are induced by mutations leading to inappropriate Hh pathway
activation. For example, loss-of-function mutations of the Patched 1 (PTC1)
gene and excessive-activation mutations of the Smoothened (SMO) gene
might cause a number of human cancers [6,12–16]. However, treatment with
specific Hh pathway inhibitors, such as KAAD-cyclopamine, can lead to growth
inhibition of cancer cells [6,9,12–16].5-FU is an important, traditional chemotherapeutic drug. Adjuvant
5-FU-based chemotherapy is widely used in the clinical treatment of many
cancers, such as stage III colon cancer [17], gastrointestinal malignancies
[18], locally advanced unresectable pancreatic cancer [19], oropharyngeal
cancer [20],
and some patients with hepatoma [21–23]. Though some
researchers have addressed the mechanism of 5-FU action [21,23], there is only
limited knowledge on the relationship between Sonic hedgehog (SHh) signaling
pathway and 5-FU. Further investigation of the function of 5-FU will help in
developing new strategies to increase its anticancer effect and will
contribute to the design of more powerful 5-FU derivatives. Previous studies have shown that SHh signaling activation is an
important event in the development of human HCC [1]. Chen et al showed
that Gli1 short interfering RNA combined with 5-FU chemotherapy may be a more
promising strategy against HCC [8].In the present study, we explored whether SHh signaling pathway
activity was involved in the 5-FU-induced inhibition of cell viability and
motility in the Hep3B cell line. Based on our previous data [1], the Hep3B cell
line is known to have typical Hh signaling pathway activity, while the HepG2
cell line does not have typical activity. Furthermore, we also determined
whether the target molecules in the SHh signaling pathway could potentially
serve as predictive biomarkers in 5-FU-based HCC chemotherapy in the future. Materials and Methods
Cell culture
Human HCC cell lines Hep3B and HepG2 were both purchased from the Cell
Bank, Chinese Academy of Sciences (Shanghai, China), and cultured in Dulbecco’s
modified Eagle’s medium (Gibco, Gaithersburg, USA), supplemented with 10 mM
HEPES (Gibco), 5 mM L-glutamine (Gibco), and 10% fetal bovine serum
(Gibco) in a humidified atmosphere of sterile air, 5% CO2 at 37 ?C.
Construction of pCS2-Gli1 expression vector and transfection
Human full-length complementary DNA from Gli1 was cleaved
from the pBluescript SK-Gli1 (a gift from Dr. Jingwu Xie, Department of
Pharmacology and Toxicology, Sealy Center for Cancer Cell Biology, University
of Texas, Galveston, USA) by HindIII and XbaI, and subcloned into
the pCS2 expression vector. Transient transfection of pCS2-Gli1 expression
vector in HCC cells was carried out using Lipofectamine 2000 reagent
(Invitrogen, Carlsbad, USA), according to the manufacturer’s recommendations. One day before transfection, 4000
cells per well were plated in a 6-well plate without antibiotics so that cells
would be 90% confluent at the time of transfection. The cells were transfected
with 4 mg plasmids, using 10 ml of Lipofectamine 2000. media were changed
at 24 h after transfection and replaced with 2 ml of fresh complete medium. To detect transfection efficiency, the co-transfection of
Gli1/green fluorescent protein (GFP) expression vectors was also carried out.
Phase contrast and fluorescence microscope microscopy
Phase contrast and fluorescence microscope microscopy
Cells were placed into 24-well plates and cultured for 24 h,
followed by transfection. As in previous reports and viability tests for IC50 at 48 h (Fig. 1) [23], the cells were treated with 150 mM 5-FU (19.51 mg/ml) every 24 h
for 3 d, observed and then photographed under a phase contrast microscope
(Nikon, Tokyo, Japan). Cells in each well were collected by spinning and
resuspended and stained with 0.5% trypan blue. Using a microscope, we counted
the number of unstained cells within limited grids in blood cell-counting
chambers. Then, the total number of living cells in each well was calculated.
Cells were fixed with 4% formaldehyde, pH 7.6, in another plate, stained with
acridine orange or Hochest33258 respectively, then cells were observed and
photographs were taken under a fluorescence microscope (Nikon).
MTT assay for cell viability
Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT; Sigma, St. Louis, USA) assay [23]. Briefly, in 96-well plates,
different groups (including a transfection group) of Hep3B and HepG2 cells were
treated for 6, 12, 24, 48, 72, 96 and 120 h with 100 ml Dulbecco’s modified Eagle’s
medium containing 0.1% dimethyl sulfoxide (DMSO), 150 mM 5-FU (19.51 mg/ml) and 2 mM
KAAD-cyclopamine respectively. About 20 ml of 5 mg/ml MTT in PBS was
added to each well including blank groups, and the plates were put into CO2 incubator at 37 ?C for 4 h. Then, when the supernatant was gently
removed, the formed formazan crystals were dissolved in 100 ml of DMSO. Using
a microplate reader (Bio-Rad, Hercules, USA), we evaluated the absorbance as
having an optical density value of 570 nm.
TUNEL assay
Hep3B cells were seeded onto sterile glass coverslips in a 6-well
plate, and then transfected. 5-FU was administered, and after 48 h, using an in
situ cell death detection kit, fluorescein was used to detect apoptotic
cells according to the recommended protocol (Roche Dignostics, Basel,
Switzerland). Terminal deoxynucleotidyl transferase fluorescence-dUTP nick end
labeling (TUNEL) assay. The percentage of TUNEL positive cells was calculated
under a fluorescent microscope. At least 500 cells were counted in each
experiment [1]. The samples were analyzed under a fluorescence microscope with
450–500
nm blue light activation and 515–565 nm green light detection.
Immunocytochemistry
The immunocytochemistry procedure was modified as follows. After the
cells were washed three times with PBS for 15 s, then fixed with 4%
paraformaldehyde in PBS (pH 8) for 10 min. And the coverslips were incubated in
primary antibody at room temperature for 1 h. After washing with PBS for three
times, the cells were incubated in second antibody for another 1 h. Immunofluorescence was carried out using the primary antibodies
(PTC1, Cat. #6149; 1:300; Santa Cruz Biotechnology, Santa Cruz, USA) and Alexa
Fluor488-tagged secondary antibodies [1], and then observed under a fluorescence
microscope (Nikon), Alexa Fluor488 is excited at 475 nm laser light.
RNA extraction and reverse transcription-polymerase chain reaction
(RT-PCR)
RNA extraction was carried out using 20 ml Trizol total RNA extraction
reagent (Tiangen, Beijing, China) according to instructions with DNase I
digestion (Promega, Madison, USA). PCR experiments were carried out in 25 ml system using 2?PCR Taq MasterMix (Tiangen). The specific primer sequences,
annealing temperature and cycles are shown in Table 1. The band
intensity of each sample was quantitatively analyzed using Quantity One
software (Bio-Rad). The RT-PCR control was carried out by amplification of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA)
using primers (Table 1).
Western blot analysis
PTC1 protein level was measured by Western blot using a PTC1
antibody (1:5000; Santa Cruz Biotechnology). Equal amounts of protein (5 mg per lane) were
loaded onto a 10% Tris-glycine gel, separated by electrophoresis, and
transferred to an Immobilon P membrane (Bio-Rad). The membranes were incubated
in blocking buffer (0.2 mM Tris, 137 mM NaCl, 5% no-fat milk, and 0.1% Tween
20) for 1 h and then probed at 4 ?C overnight with PTC1 antibody (1:5000;
Santa Cruz Biotechnology). The membranes were rinsed with washing buffer (0.1%
Tween 20, 0.2 mM Tris, and 137 mM NaCl) and incubated with horseradish
peroxidase-conjugated secondary antibody (1:5000) for 1 h at room temperature,
which was followed by chemiluminescent detection (Pierce, Rockford, USA).
Scratch wound closure assay for motility
Hep3B cells (3104/well) were seeded in a 6-well
plate and grown to 80% confluency before transfection. After 24 h, the monolayer
was scratched with a pipette tip to create a cell-free strip area [24–26], gently
washed with PBS to remove floating cells and photographed at three random
locations (0 h). We set up different wells for control (0.1% DMSO solution)
and for 5-FU-treated groups with and without transfection. A photograph of cell
migration was taken with a microscope (Nikon) every 24 h for 3 d at the same
locations.
Statistical analysis
All data were expressed as mean±SE. Student’s t-test with
Welch’s correction was used to evaluate the differences. Differences between
two groups were determined using P-values. P<0.05 was considered statistically significant with marker *. All experiments were repeated three times.
Results
Expressions of SHh pathway target genes in Hep3B cells treated with
5-FU were down -regulated
It has been reported that the Hep3B cell line has SHh signaling
pathway activity but that HepG2 does not, though the pathway ligand SHh could
be detected [1]. To determine whether 5-FU treatment could affect SHh pathway
activity, we examined the expression of SHh pathway related genes by RT-PCR.
The genes studied included ligand SHh gene, receptor SMO gene,
receptor PTC1 gene (a target gene in the SHh pathway), and the
transcript factor Gli1 gene, which could regulate the transcription of
target genes in SHh pathway. The expressions of Gli1 and PTC1
were believed to indicate the pathway activation [6,27]. We have also detected
the gene expression of Su(Fu) and HIP, both of which are negative
regulators of the SHh pathway. Our results revealed that 5-FU in Hep3B cells
could distinctly decrease the expression of PTC1 and Gli1 genes;
the mRNA levels of SHh, SMO and Su(Fu) were also
down-regulated, but HIP was not affected [Fig. 1(A)]. As in
previous reports [1,6], neither PTC1 nor Gli1 mRNA was detected
in HepG2 cells, and SHh expression was not affected [Fig. 1(B)]. To
confirm the above results, Western blot was carried out using PTC1 antibody.
The parallel result was obtained at protein level [Fig. 1(D)].
Overexpression of Gli1 could rescue the inhibition of cell
proliferation caused by 5-FU
To determine whether SHh pathway activity is involved in the
inhibition of cell proliferation caused by 5-FU, an expression plasmid,
pCS2-Gli1, was constructed and transfected into Hep3B cells. To avoid
interfering with Gli1 function, no tag was added in this construction. The
transfection efficiency was over 30%, as shown by co-transfection of pCS2-GFP
in the same system [Fig. 2(A)], and was confirmed by semi-quantitative
RT-PCR [Fig. 2(B)]. 5-FU was added to the medium 48 h after transfection
and time-lapse MTT assay was carried out. The results revealed that the
experiment groups (Gli1 plasmid transfection combined with 5-FU treatment)
showed a higher growth rate than the 5-FU-treated groups (Fig. 3). They
also indicated that the overexpression of Gli1 could rescue cell proliferation
repressed by 5-FU. To determine whether the rescue effect was caused by the
inhibition of apoptosis in Hep3B cells, TUNEL assays were performed. As shown
in Fig. 4, 5-FU treated groups showed evident apoptosis, particularly
when compared with the control (P<0.05). However, Hep3B cells overexpressing Gli1 showed a lower rate of apoptosis compared with the 5-FU treated groups (P<0.05), as confirmed by phase contrast microscope (data not shown). These results were also confirmed by Hochest33258 fluorescent staining (Fig. 5) and acridine
orange staining (Fig. 6). The rescue ability of the Gli1 plasmid
transfection group was also evident (P<0.05). Additionally, the repression extent by 5-FU is deeper than that by KAAD-cyclopamine, a specific inhibitor of Hh pathway; this may have resulted from the presumption that the down-regulation of the SHh pathway partially accounts for the antitumor effect of 5-FU. To get more direct evidence, we employed one of most credible experiments for testing cell viability, cell counting assay with trypan blue dyeing, using a hemocytometer on Hep3B cells (Fig. 7) [28–30]. The results
are in accordance with those of MTT.
Overexpression of Gli1 could restore the cell motility
repressed by 5-FU
We detected the influence of the SHh signaling pathway on the
motility of Hep3B cells treated with 5-FU in scratch wound closure assay.
Higher mobility was observed in the group treated with 0.1% DMSO. The retrieval
effect on cell migration occurred in the 5-FU-treated Hep3B cells with Gli1
overexpressed [Fig. 8(A)]. In contrast, the 5-FU-treated HepG2 cells
showed few changes [Fig. 8(B)]. For Hep3B cells, the control group
showed marked migration after 24 h, while the 5-FU-treated groups did not.
Despite 5-FU treatment, the transfection groups still showed significant
motility after 48 h, compared with the initial morph of wound closure. For
HepG2 cells, 5-FU seemed less effective on cell motility than Hep3B.
The subcellular location of PTC1 protein changed in 5-FU-treated
cells
To investigate how 5-FU disturbed the SHh pathway signaling in
vitro, immunofluorescent assay was performed to detect the subcellular
location of PTC1 protein. In the control group, PTC1 protein appeared markedly
and mainly on the cell membrane and partially around the nucleus in the
cytoplasm (Fig. 9). When treated with 5-FU, PTC1 protein expression was
evidently down-regulated, and nearly all the PTC1 proteins were dispersed
across the cytoplasm. Furthermore, in the transfection group, the expression of
PTC1 protein was restored by the overexpression of Gli1. Analysis by fluorescent
intensity was carried out to confirm the differences at 0, 24 and 48 h. The
expression level of PTC1 protein in the 5-FU-treated groups was dramatically
reduced after 48 h treatment.
Discussion
More than 600,000 cases of liver cancer, mostly HCC, are diagnosed
globally each year. Because the majority of HCC cases are advanced or involve
an unresectable malignancy, it is characterized as a highly chemoresistant
cancer with no effective systemic therapy [1]. It
is the utmost important for us to gain a better understanding of the molecular
mechanism of HCC development and to find more effective therapeutic approaches.
Several important intracellular signaling pathways, such as Ras,
phosphatidylinositol-3 kinase, Wnt and Hh (the latter two are related to
embryogenesis), have been shown to function in HCC [9,31,32]. The role of the
Hh pathway in human liver cancer was established by Sicklick et al and
by our previous study [1,2]. Many investigations have indicated that targeted
inhibition of the Hh pathway in Hh-activated cancer cell lines results in
growth inhibition [6,9,13,14,16]. Our findings revealed that the expression of
SHh pathway target genes was decreased in Hep3B cells treated with 5-FU. The
data also indicated that overexpression of Gli1, a direct
transcriptional Hh target gene, could restore the proliferation ability and
cell motility suppressed by 5-FU in Hep3B cells. It implied that the
down-regulation of SHh signaling pathway activity was involved in cancer cells
5-FU-induced apoptosis. Our results were consistent with the recent report by
Chen et al that found the viability of Huh7 cells reduced when treated
with Gli1 short interfering RNA and 5-FU [8]. We also found that PTC1 had an abnormal subcellular location in
Hep3B cells treated with 5-FU. It suggested that 5-FU might directly or
indirectly change the function of PTC1 protein in Hep3B cells. PTC1 is an
important part of the Hh signaling pathway and serves as the receptor for the
secreted Hh molecule. The failure of PTC1-mediated suppression of SMO triggered
a cascade of intracellular events and activated the SHh signaling pathway [33].
It has been established that loss-of-function mutation in PTC1 occurs
frequently in human basal cell carcinomas and medulloblastomas [34]. However,
PTC1 might relate to the regulation of cell division [35]. Our data suggested
that 5-FU might down-regulate SHh signaling pathway activity through a
malfunction of PTC1, which subsequently induces the apoptosis and the
inhibition of motility of Hep3B cells, a classic Hh-activated HCC cell line.In summary, our results indicated that down-regulation of SHh
signaling pathway activity was involved in 5-FU antitumor effect. Thus,
5-FU-based combination chemotherapy that inhibits the SHh signaling pathway
might represent a promising strategy against HCC. To develop prospective
therapeutic strategies of HCC, key marker genes that respond to 5-FU must be
defined. As such, further investigation in multiple HCC cell lines and in
vivo should be conducted to benefit preclinical prevention and potential
clinical application in the future.
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