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Introduction
Pancreatic carcinoma is characterized by its aggressive local invasion of adjacent structures. At the time of first diagnosis,
only 10%_20% of cases are eligible for the potentially curative Whipple's procedure. Furthermore, pancreatic cancer is
relatively resistant to both chemotherapy and
radiotherapy[1]. Further understanding of the biological roles of the genotypic
changes during pancreatic carcinogenesis may provide new clues for developing strategies to prevent and treat this disease.
Among the various genotypic changes occurring in pancreatic cancer, the dysregulation of the sonic hedgehog (SHH)
signaling pathway has been reported[2]. The SHH protein is expressed in an embryo and participates in regulating cell
proliferation, differentiation, and tissue patterning of many organs, including pancreas. The SHH ligand mediates its
biological effects through the multi-component receptor complex constituted from a transmembrane protein PATCHED-1 (PTCH-1).
PTCH-1 binds SHH with high affinity and Smoothened (SMO), a second signaling transmembrane G protein-coupled receptor.
In the absence of SHH, PTCH-1 represses SMO activity,
while the binding of SHH to PTCH-1 releases the basal
repression of SMO by PTCH-1. This ultimately leads to the
activation of the glioma-associated oncogene homolog
1(GLI-1) transcription factor, which induces the expression of
numerous target genes that regulate proliferation, differentiation,
and extracellular matrix
interactions[3]. Recently, this signaling pathway has been
shown to have some interactions with other pathways, such
as the PI3-kinase/Akt signaling
pathways[4], and plays a major role in several types of gastrointestinal
cancers[5]. The abnormal activation of the SHH pathway in carcinogenesis
makes it a potential target for therapy. Another pathway
that may play an important role in pancreatic carcinogenesis
is the family members of epidermal growth factor
receptor(EGFR) and their ligand molecules. The overexpression of
EGFR signaling increases the proliferation of pancreatic
cancer cells[6].
While the expression of SHH and EGFR has been
reported in many tumors, respectively, the relationship
between these signaling pathways was unclear. Moreover,
few studies have been reported about the co-expression and
simultaneous inhibition of SHH and EGFR signaling in
pancreatic cancer cells. The aim of the present study was to
detect the elements of the SHH and EGFR pathways in 3
pancreatic cancer cell lines, and to determine whether the
simultaneous inhibition of SHH and EGFR pathways might
be more effective than single agents. Hence, the
antiprolifera-tive effects induced by cyclopamine, a SHH signaling
specific inhibitor, alone or in combination with EGFR inhibitor
Iressa, were investigated in the PANC-1, SUIT-2, and
ASPC-1 pancreatic cancer cell lines, and the interaction between
these 2 pathways was evaluated.
Materials and methods
Cell culture Pancreatic cancer cell lines (PANC-1,
SUIT-2, and ASPC-1) were obtained from the Chinese Academy of
Medical Science and Peking Union Medical College (Beijing,
China), generously provided by Dr Hai HU. SUIT-2 was
moderately differentiated, ASPC-1 was moderately to poorly
differentiated, and PANC-1 was poorly
differentiated[7]. All the cells were grown in a monolayer culture in a humidified
atmosphere with 5% CO2 and 95% air at 37 °C. ASPC-1 and
SUIT-2 cells were grown in RPMI-1640 (Sigma Chemical Co,
St Louis, MO, USA) medium and PANC-1 cells in Dulbecco's
modified Eagle's medium (Sigma Chemical Co, St Louis, MO,
USA), respectively, supplemented with 10% fetal bovine
serum, 100 U/mL penicillin, and 100 mg/mL streptomycin.
RNA isolation and RT-PCR The transcription levels of
SHH, SMO, and EGFR in the pancreatic cancer cells were
detected by RT-PCR analyses before and after the addition
of cyclopamine and Iressa, alone or in combination. The
pancreatic cancer cells were collected by centrifugation and
the total RNA was isolated from the cultures using Trizol
reagent (Qiagen, Hilden, Germany). After quantification,
mRNA was transcribed into first-strand cDNA using the
Avian Myeloblastosis Virus Reverse Transcriptase
(AMV-RT) (Promega Biotech Co, Madison, WI, USA).
Conventional PCR reactions were carried out in a total volume of 20
µL containing 1× PCR-buffer (Toyobo Co., Kita-ku, Osaka
City, Japan), 150 µmol/L of each nucleotide, 20 pmol/L of
each SHH, SMO, or EGFR primer, and 1 U of
Taq polymerase (Toyobo, Co., Kita-ku, Osaka City, Japan). The PCR
products were separated by agarose gel electrophoresis and
visualized by ethidium bromide staining.
The following primer sequences were employed:
SHH (170 bp): sense 5'-GAAAGCAGAGAACTCGGTGG-3' and antisense 5'-GGAAAGTGAGGAAGTCGCTG)-3'; SMO
(263 bp): sense 5'-ATCTCCACAGGAGAGACTGGTTCGG-3' and antisense 5'-AAAGTGGGCCTTGGGAACATG-3';
EGFR (194 bp): sense 5'-GTGGCTGGACTGCTCAAGAG-3' and antisense 5'-CTAGTCTCGAGTAGGCCTTTGTG-3';
β-actin (410 bp): sense 5'-CTACGAGCTGCCTGACG-3' and
antisense 5'-AGAAGCATTTGCGGTGG-3'.
Western blot analysis The pancreatic cancer cells were
solubilized in lysis buffer containing 50 mmol/L Tris-HCl, 1%
Nonidet P-40 (NP-40), 0.25% Na-deoxycholate, 150 mmol/L
NaCl, 1 mmol/L EDTA, 1 mmol/L phenylmethanesulfonyl
fluoride(PMSF), and the protease inhibitors of the cocktail
(Roche Diagnostics Ltd, Basel, Switzerland). The lysate were
centrifuged at 10 000×g for 30 min to remove insoluble material.
The total protein concentration was calculated according to
the Bradford protein quantification method. Samples of equal
amount were loaded onto a 10% SDS-PAGE gel and
electrophoretically transferred to a Polyvinylidene
Difluoride(PVDF) membrane. After blocking with 5% non-fat milk/
Tris-Tween buffer saline (TTBS), the membrane was
incubated for 90 min with a highly specific primary antibody of
anti-EGFR (sc-03, Santa Cruz Biotechnology, Santa Cruz,
CA, USA) and anti-SHH (sc-1194, Santa Cruz
Biotechno-logy, Santa Cruz, CA, USA), respectively, then washed with
0.05% Tween-20 in Tris buffer saline TBS and incubated with
horseradish peroxidase-coupled secondary antibody for 60
min. Protein signals were detected using an
electrochemilumi-nescence(ECL) reagent (Amersham Pharmacia Biotech,
Buckingham, UK).
Iressa and cyclopamine treatment The
logarithmically-growing pancreatic cancer cells were plated at a density of
1×104 cells/well into a 96-well plate, followed by 24 h of
serum starvation to synchronize. Cyclopamine (Toronto
Research Chemicals, Ontario, Canada) of different
concentra-tions, alone or in combination with Iressa (Astra Zeneca
Pharmaceuticals LP, Wilmington, DE and Cheshire, UK), were
added to the culture medium. Iressa treatment was performed
at 3 doses (1, 2.5, and 5 µmol/L, respectively), whereas
cyclopamine treatment was performed at 2 dosages (2.5 and
5 µmol/L, respectively). DMSO was used at a final
concentration of 0.1% in the control wells. After treatment for 72
h, the rate of cell growth and apoptosis was measured.
Methyl thiazolyl tetrazolium analysis 20 µL methyl
thiazolyl tetrazolium (MTT) (5 g/L) was added to each well
and incubated for an additional 4 h; the culture media were
then discarded, followed by the addition of 0.15 mL DMSO,
and then vibrated for 10 min. The absorbance was measured
at 490 nm using a model 550 microplate reader. The
inhibitory rates (IR) were calculated as follows: IR
(%)=([1_absorbance of the treated wells]/[Absorbance of the control
wells])×100.
Flow cytometry analysis The pancreatic cancer cell
density was adjusted to
(0.3_1.0)×107 cells/mL. The cells were
serum starved for 24 h and then treated with different
concentrations (2.5 or 5 µmol/L, respectively) of cyclopamine,
alone or in combination with 1 µmol/L Iressa for 72 h. Then
the cells were harvested with trypsin-EDTA to produce a
single cell suspension. The cells were pelleted by
centrifugation and washed twice with phosphate-buffered solution
(PBS). Then the cell pellets were resuspended in 0.5 mL PBS
and fixed in 5 mL ice-cold 70% ethanol at 4 °C. The fixed cells
were spun down by centrifugation and the pellets were
washed with PBS. After being resuspended in 1 mL PBS, the
cells were incubated with RNase A (20 mg/L, Sigma Chemical
Co, St Louis, MO, USA) and propidium iodide (PI) (50 mg/L,
Sigma Chemical Co, St Louis, MO, USA) and shaken for 1 h
at 37 °C in the dark. The stained cells were analyzed using a
FACScan flow cytometer in combination with BD analysis II
software (Becton Dickinson, Franklin Lakes, NJ , USA). The
apoptosis rates were calculated.
Statistical analysis Data were expressed as mean±SD.
The data were analyzed using one- or two-way ANOVA
according to the experimental design applied. The simple and
the repeated contrasts were applied to ANOVA in order to
compare the various experiments with the control experiment
as well as adjacent categories, respectively. All statistical
analyses were performed using the SPSS 11.0 software
package for Windows (SPSS, Chicago, IL, USA). A two-tailed
P-value less than 0.05 was considered statistically significant.
Results
mRNA transcription levels of SHH, SMO, and EGFR
SHH, SMO and EGFR mRNA were transcribed in all of the 3
pancreatic cancer cell lines. The PANC-1 cells exhibited weak
transcription, whereas the other 2 cell lines were found to
have a high transcription level of SHH, SMO, and EGFR at
the mRNA level (Figure 1).
Detection of SHH and EGFR protein expressions by
Western blot analysis The supernatant of the cultured cells
in the 3 groups was collected and analyzed by Western
blotting. The SHH and EGFR protein expressions were found
in all the 3 cell lines, and the expression level of the SHH and
EGFR proteins were lower in PANC-1 than in ASPC-1 and
SUIT-2 (Figure 2).
Effect of cyclopamine on the expression of EGFR
To identify the relationship between the SHH and EGFR
signaling pathway, the effect of cyclopamine on the expression of
EGFR was investigated. After exposure to cyclopamine (5
µmol/L) for 48 h, RT-PCR and Western blot analysis were
carried out and revealed that cyclopamine could significantly
suppress the transcription and expression levels of EGFR
(Figures 3, 4).
Effect of cyclopamine and Iressa on cell
proliferation A MTT assay was used to investigate the cell viability of
PANC-1, ASPC-1, and SUIT-2 after cyclopamine and Iressa
treatment. As shown in Figure 5, cyclopamine could inhibit
the growth of the pancreatic cancer cells in a
dose-dependent manner. SUIT-2 cells demonstrated the strongest
response among the 3 cell lines due to the overexpression of
SHH signaling. Even at a minimum dose of 2.5 µmol/L
cyclopamine, SUIT-2 cells showed a significant
(P<0.001) growth reduction (81.6%), whereas PANC-1 and ASPC-1 cells
showed a moderate response (51.4% and P=0.047 and 60.8%
and P=0.035, respectively). At the dose of 5 µmol/L, almost
complete growth inhibition (91.7% reduction) was observed
in SUIT-2 cells. At this dose, PANC-1 and ASPC-1 also
showed significant reduction in growth (71.9%
and P=0.032 and 76.6% and P=0.016, respectively). The EGFR tyrosine
kinase inhibitor Iressa also induced a growth inhibitory
effect in a dose-dependent manner. The most significant
impact on growth inhibition was observed in SUIT-2 cells,
followed by ASPC-1, whereas PANC-1 demonstrated a
moderate response. At the dose of 5 µmol/L, almost complete (89.3%)
growth reduction was observed in SUIT-2 cells, a 78.2%
reduction was observed in ASPC-1 cells, and a 70.6%
reduction was seen in PANC-1 cells (P<0.01), respectively.
More-over, the combined use of 2.5 µmol/L cyclopamine and
1 µmol/L Iressa induced an enhanced inhibitory effect, which
was much more than that of 5 µmol/L cyclopamine or 5
µmol/L Iressa alone.
Effects of cyclopamine and Iressa on cell cycle
progression In order to examine the effects of cyclopamine and/or
Iressa on cell cycle progression, the pancreatic cancer cells
were treated with the indicated concentrations of cyclopamine
(2.5 and 5 µmol/L, respectively) and Iressa (1, 2.5, and 5
µmol/L, respectively), alone or in combination for 72 h. The
percentage of cells in each phase of the cell cycle,
G0/G1, S, and G2/M was determined by flow cytometry after the cells
were treated. As shown in Table 1, the percentage of the cell
population of the G0/G1 phase was significantly increased,
while that in S and G2/M phases decreased. Cyclopamine
and/or Iressa caused cell cycle arrest in the
G0/G1 phase in a dose-dependent manner.
Effect of cyclopamine and Iressa on
apoptosis After the pancreatic cancer cells were exposed to the indicated
concentrations of cyclopamine (2.5 µmol/L) and Iressa (1.0
µmol/L), alone or in combination for 72 h, a flow cytometry analysis
was carried out and revealed that the apoptotic rate of
PANC-1 was 18.17%±0.73%, 11.82%±0.24%, and
32.55%± 0.56%, respectively. The apoptotic rate in the control group was
1.38%± 0.45% and the apoptotic rate of SUIT-2 was higher than the
other 2 cell lines. Cyclopamine and Iressa induced apoptosis
of the pancreatic cancer cells in a dose-dependent manner.
Moreover, 2.5 µmol/L cyclopamine plus 1 µmol/L Iressa was
more effective to all tested pancreatic cancer cells than agents
alone and this combination caused the death of the majority
of cells (Table 2).
Discussion
SHH signaling is important for normal axial patterning of
the mammalian embryo and has been proved to be essential
for foregut development. Recently, some studies have
explored the role of inappropriate activation of the SHH
signaling pathway in several types of gastrointestinal
tumors[5]. On the other hand, EGFR could promote proliferation and
differentiation of some epithelial cells both in
vitro and in vivo[8]. EGFR has been involved in the development of many
tumors, such as lung, breast, prostate, and colon
cancers[9]. In our study, the overexpression of endogenous SHH ligands,
SMO, and EGFR were detected by RT-PCR and Western blot
analysis in pancreatic cancer cell lines. However, the
expression level of these molecules was different in these cells.
SUIT-2 cells have the highest expression level, whereas
PANC-1 cells have the lowest. The concomitant elevated
expression of SHH and EGFR appears to be implicated in the
activation of mitotic signaling[10,11], resulting in a
continuance proliferation of pancreatic cancer cells.
In our studies, when the pancreatic cancer cells were
treated with cyclopamine, the mRNA and protein levels of
EGFR decreased accordingly, which means that the
cyclopa-mine treatment of pancreatic cancer cells could downregulate
the expression of EGFR. It has been reported that the SHH
ligand could induce the growth of pancreatic cancer cells in
an autocrine fashion[12]. The stimulation of the SHH ligand
has been reported to increase Drosophila
EGFR (DER) signaling by upregulating the expression of the
EGFR[13]. Furthermore, it has been reported that the aberrant
activation of SHH signaling in the precursor cells results in the
initiation of some tumors, such as brain and
skin[14,15].
Although it remains uncertain as to whether or not these
tumors come from the aberrant activation of SHH and EGFR
in stem cells or differentiated cells, it seems that there must
be some interaction between these signaling
pathways[10,16,17].
As an inhibitor of the SHH signaling pathway, cyclopamine is an alkaloid isolated from the
Veratrum californicum plant[18]. Berman
et al found that treatment with cyclopamine inhibited the growth of digestive tract
cancer significantly[5]. The inhibitory role of cyclopamine is being
tested for other cancers as well, for example
medulloblastoma[19],
breast[20], and prostate
cancers[10,21,22]. In our studies, it was demonstrated that cyclopamine could induce the
inhibition in pancreatic cancer cells with activated SHH signaling,
but different cell lines present variable reactions to
cyclopamine. For SUIT-2 cells, treatment with 2.5 µmol/L
cyclopamine induced an inhibitory rate of 81.6%, whereas
the rate of PANC-1 and ASPC-1 cells was 51.4% and 60.8%,
respectively. This difference might be due to the different
expression of SHH in different cell lines. Perhaps other
genetic alterations, such as the Indian Hedgehog (IHH) and
EGFR signaling pathways, may contribute to it. Recently, a
report indicated that IHH, another member of the HH family,
may be the dominant ligand expressed in both pancreatic
cancer and chronic pancreatitis[23]. Further understanding
of the molecular basis for cell sensitivity to cyclopamine will
help us design better ways to manipulate pancreatic cancer
in the future. Thus, it may be possible in the future to treat
the subsets of pancreatic cancer with SHH signaling
inhibitors.
It has been reported that the blockade of the EGFR
signaling pathway with Iressa, a selective EGFR inhibitor,
resulted in antiproliferative activity in human cancer cell lines
of different histological types, such as ovarian, breast, and
colon cancers[24]. Our results indicated that Iressa also
inhibited the proliferation of pancreatic cancer cells in a
dose-dependent manner. Moreover, the combination of 2.5 µmol/L
cyclopamine and 1.0 µmol/L Iressa resulted in a higher
inhibitory effect in pancreatic cancer cells than any single
agent. Hence, these pathways might act cooperatively for
the proliferation of pancreatic cancer cells. In our study, the
pancreatic cancer cells were arrested in the
G0/G1 phase after cyclopamine treatment, concomitant with a decrease of the
cell number in the S and G2/M phases. It has been reported
that the SHH signaling could be activated by the upregulation
of cell cycle regulators, such as
cyclins[25]. Therefore, cyclopamine could downregulate the expression of cell cycle
regulators through the inhibition of SHH signaling, and as a
result, the mitogenic effect induced by other growth factors,
such as EGF, might be balanced out[10].
In this study, the apoptotic effect induced by
cyclo-pamine, alone or at lower concentrations with Iressa in
pancreatic cancer cells was also investigated. Cyclopamine and
Iressa induced apoptosis of pancreatic cancer cells in a
dose-dependent manner. Moreover, 2.5 µmol/L cyclopamine plus
1 µmol/L Iressa caused a stronger inhibitory effect to all
tested pancreatic cancer cells than agents alone and this
combination caused the death of the majority of cells.
Further studies of SHH signaling in different stages of
pancreatic cancers, especially at early stages, will facilitate
early diagnosis of pancreatic cancer through the detection
of SHH signaling. Moreover, the simultaneous blockade of
the SHH and EGFR signaling pathways leads to an arrest of
growth and an increase of apoptosis in pancreatic cancer
cells. These pathways may offer an exciting, new
therapeutic approach for pancreatic carcinoma which is still treated
by palliative procedures or highly toxic drugs. The selective
inhibition of signaling pathways activated in precancerous
cells should be possible without disturbing other pathways
necessary for normal cell function, also minimizing toxicity
from such therapy. Cyclopamine appeared to selectively
induce apoptosis in tumor cells without adverse effects on
normal tissues in vivo[26], thus, it was an ideal drug with
potential to prevent the progression of pancreatic cancer
cells.
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