Cyclopamine reverts acquired chemoresistance and down-regulates cancer stem cell markers in pancreatic cancer cell lines.

Abstract

The hedgehog (Hh) pathway has been implicated in the pathogenesis of cancer including pancreatic ductal adenocarcinoma (PDAC). Recent studies have suggested that Hh plays an important role in maintaining the cancer stem cell (CSCs) pool. Gemcitabine-resistant pancreatic cancer cells highly express some of the CSCs markers. However, the expression level of Hh members in gemcitabine-resistant pancreatic cancer cells remains unknown. The aim of this study was to verify the expression of HH members, such as Shh, Ptc, SMO and Gli-1 in gemcitabine-resistant PDAC cell lines, and to explore a new strategy to overcome chemoresistance in PDAC.
Quantitative real-time RT-PCR (Q-PCR) and western blot were used to evaluate the relative expression level of HH members in SW1990, CFPAC-1 cells and gemcitabine-resistant SW1990, CFPAC-1 cells. The change of cancer stem cell markers and the expression level of HH members before and after cyclopamine treatment was evaluated using flow cytometry and Q-PCR, western blot, respectively. Cell apoptosis after cyclopamine treatment was measured by flow cytometry.
CD44, CD133 and the expression level of HH members, including Shh, SMO, Gli-1, were found to be highly expressed in gemcitabine-resistant cells, which were significantly down-regulated by cyclopamine treatment. Flow cytometry analysis showed increased cell apoptosis after cyclopamine treatment.
Gemcitabine-resistant pancreatic cancer cells highly express CSCs markers and some of the HH members, and inhibition of HH by cyclopamine is an effective method of reversing gemcitabine resistance in pancreatic cancer.

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Basic science in medicine | Published 31 May 2011, doi:10.4414/smw.2011.13208
Cite this as: Swiss Med Wkly. 2011;141:w13208
Cyclopamine reverts acquired chemoresistance
and down-regulates cancer stem cell markers in
pancreatic cancer cell lines
Jie Yaoa, 1, Yong Anb, 1, Ji-shu Weib, Zhen-ling Jic, Zi-peng Lub, Jun-li Wub, Kui-rong Jiangb, Ping Chena, Ze-kuan Xub, Yi Miaob
aDepartment of General Surgery, The First Affiliated Hospital of Yangzhou University, Yangzhou 225001, P. R China
bCentre for Pancreatic Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210009, P. R China
cDepartment of General Surgery, Zhongda Hospital, Southeast University, Nanjing, 210009, P. R China
1These authors contributed equally to the study
Summary
BACKGROUND: The hedgehog (Hh) pathway has been
implicated in the pathogenesis of cancer including pancre-
atic ductal adenocarcinoma (PDAC). Recent studies have
suggested that Hh plays an important role in maintaining
the cancer stem cell (CSCs) pool. Gemcitabine-resistant
pancreatic cancer cells highly express some of the CSCs
markers. However, the expression level of Hh members in
gemcitabine-resistant pancreatic cancer cells remains un-
known. The aim of this study was to verify the expression
of HH members, such as Shh, Ptc, SMO and Gli-1 in
gemcitabine-resistant PDAC cell lines, and to explore a
new strategy to overcome chemoresistance in PDAC.
MATERIAL AND METHODS: Quantitative real-time RT-
PCR (Q-PCR) and western blot were used to evaluate the
relative expression level of HH members in SW1990,
CFPAC-1cellsandgemcitabine-resistant
CFPAC-1 cells. The change of cancer stem cell markers
and the expression level of HH members before and after
cyclopamine treatment was evaluated using flow cytometry
and Q-PCR, western blot, respectively. Cell apoptosis after
cyclopamine treatment was measured by flow cytometry.
RESULTS: CD44, CD133 and the expression level of HH
members, including Shh, SMO, Gli-1, were found to be
highly expressed in gemcitabine-resistant cells, which were
significantly down-regulated by cyclopamine treatment.
Flow cytometry analysis showed increased cell apoptosis
after cyclopamine treatment.
CONCLUSION: Gemcitabine-resistant pancreatic cancer
cells highly express CSCs markers and some of the HH
members, and inhibition of HH by cyclopamine is an ef-
fective method of reversing gemcitabine resistance in pan-
creatic cancer.
SW1990,
Key words: cancer stem cells; hedgehog signaling
pathway; gemcitabine; drug resistance; cyclopamine;
pancreatic cancer; chemoresistance
Introduction
Pancreatic cancer is among the most devastating of human
malignancies. Despite recent improvements in surgical and
chemotherapeutic approaches, pancreatic cancer continues
to have a dismal prognosis, with an average overall median
survival of 4–6 months. Overall 5-year survival is less than
5% [1]. Currently, gemcitabine is the standard chemothera-
peutic agent used in patients with pancreatic cancer [2].
However, the clinical impact of gemcitabine remains mod-
est [3]. This limitation in conventional treatments is mainly
due to the profound resistance of cancer cells to anti-cancer
drugs, which can be inherent and/or acquired [4, 5]. To
study acquired chemoresistance in pancreatic cancer, sev-
eral drug-resistant pancreatic cancer cell lines have been
established. In the research of Shah [6] and Hong [7], pan-
creatic cancer stem cells (CSCs) had been found to be en-
riched in gemcitabine-resistant pancreatic cancer cell lines.
The CSC hypothesis offers new insight into the mechanism
of drug resistance [8]. Most conventional therapies kill
most of the tumour population, but CSCs, which have in-
trinsic detoxifying mechanisms, can easily elude conven-
tional therapies. The CSCs model also explains why stand-
ard chemotherapy may result in tumour shrinkage but is
then followed by tumour recurrence and multidrug resist-
ance. Therapies targeting cancer stem cells may contribute
a new strategy for overcoming drug resistance.
Pancreatic cancer stem cells are highly tumorigenic and
possess the abilities to self-renew and produce differenti-
ated progeny. They are defined by the expression of the
cell surface markers CD44, CD24 and ESA [9] and CD133.
[10, 11]. Cancer stem cells are different from cancer cells
in many ways. Not only are cancer stem cells more res-
istant to standard chemotherapy drugs, they also employ
different signalling pathways [12]. Misregulated Hedgehog
(HH) signalling, which is normally an essential pathway
during embryonic pancreatic development, has been im-
plicated in several forms of cancer, including human pan-
creatic carcinoma [13–15]. Activation of HH signalling is
typically initiated by the binding of hedgehog ligands (Son-
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ic, India, Desert hedgehog) to a 12-transmembrane pro-
tein receptor patched (Ptc). Binding of HH to Ptc relieves
Smoothened (SMO), a 7-transmembrane protein, from the
inhibitory effect of Ptc, and activated SMO in turn triggers
a series of intracellular events, resulting in the regulation
of downstream target genes through the GLI transcriptional
effectors Gli-1, Gli-2 and Gli-3 [16, 17]. Blockage of HH
signalling has been shown to inhibit pancreatic cancer cell
growth [15], invasion, and metastases [18]. Recent studies
demonstrated that a HH inhibitor can restore drug resist-
ance in CD34+ leukaemic cells [19]. The combined block-
ade of Hh and mTOR signalling together with chemother-
apy is capable of eliminating pancreatic CSCs [11]. Lee et
al. showed that the HH signalling pathway is overexpressed
in CD44+CD24+ESA+ pancreatic cancer stem cells [9].
Gemcitabine-resistant pancreatic cells are enriched with
cancer stem cells, but whether these cells overexpress the
HH signalling pathway remains unknown.
In a previous study we established gemcitabine-resistant
pancreatic cell lines CFPAC-1/res and SW1990/res, and we
also found that CD44, ABCG2 and ABCB1 were highly
expressed in gemcitabine-resistant cells, as reported by
Hong [7]. In the present study we evaluated the expression
of the HH signalling pathway in drug-resistant pancreatic
cell lines as compared to parental cells. We also blocked
the HH signalling pathway with cyclopamine to determine
whether ABCG2, ABCB1, CD44, and CD133 can be
down-regulated in gemcitabine-resistant cells and thus
whether acquired gemcitabine-resistance can be reversed
by blockade of the HH signalling pathway.
Materials and methods
Cell culture
The human pancreatic cancer cell lines CFPAC-1 and
SW1990 were purchased from Shanghai Cell Bank (Shang-
hai, China) and cultured in Dulbecco’s modified Eagle’s
medium (DMEM) with 10% foetal bovine serum (FBS) at
Figure 1
Gemcitabine-resistant pancreatic cancer cells highly express
ABCB1 and ABCG2. A. Quantitative RT-PCR was used to
evaluate the relative expression level of ABCB1 and ABCG2 gene
expression in gemcitabine-resistant cells when compared to their
parental cells. Fold change was analysed by the 2–ΔΔCtmethod,
while the expression of β-actin was used to normalise the relative
expression of each gene within each cell line. B. The results of the
western blot analysis were consistent with the mRNA expression.
Values represent mean ± S.D. of three independent experiments.
37ºC with 5% CO2, supplemented by 1% penicillin/strep-
tomycin.
Establishment of gemcitabine-resistant pancreatic
cancer cells
Pancreatic CFPAC-1 and SW1990 cell lines were estab-
lished from spleen metastases and liver metastases of
PDAC respectively. Resistant cells were obtained by cul-
turing parental CFPAC-1 and SW1990 cells in serially in-
creasing concentrations of gemcitabine. In brief, the cells
were first cultured in medium with increasing concentra-
tions of gemcitabine, starting at the IC50, for 3 days, fol-
lowed by recovery periods in drug-free medium until the
cells regained exponential growth. The new IC50 of
gemcitabine-treated cells was then evaluated by MTT as-
say. The concentration of gemcitabine was then increased
to the new IC50to kill half of the cells. Gemcitabine was
purchased from the Lilly Company (Lilly France).
Drug sensitivity assay
To detect the IC50of both pancreatic cancer cell lines, ali-
quots of 2×103CFPAC-1 and SW1990 cells were seeded in
96-well plates with appropriate growth medium at 200 μL
per well. After a 12-h recovery period, triplicate wells were
exposed to various concentrations of gemcitabine for 72
h. A 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoli-
um bromide (MTT) assay was used to examine the effects
on cell growth. In brief, 20 μL MTT solution (5 mg/ mL)
(Sigma) was added into each well and incubated at 37 ºC
for 4 hr. A 150-μL aliquot of DMSO was then added and
absorbance was measured by a microplate reader (Mult-
iskan MK3, Thermo Labsystems, USA) at a wavelength of
Figure 2
Gemcitabine-resistant pancreatic cell lines highly express
hedgehog members. A, Quantitative RT-PCR was used to
evaluate the relative expression level of Shh, Ptc, SMO, and Gli-1
in gemcitabine-resistant cells in comparison to their parental cells.
Fold change was analyzed using the 2–ΔΔCtmethod, while the
expression of β-actin was used to normalise the relative expression
of each gene within each cell line. Values represent mean ± S.D. of
three independent experiments. B, mRNA expression of SMO can
be detected in SW1990/res cells but not parental cells. C, The
different protein expression levels of SMO and Gli-1 are consistent
with mRNA expression.
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490 nm. The cell survival rate (SR) was calculated using
the formula SR = (mean absorbance of the test well/mean
absorbance of the control) × 100%; the inhibition rate (IR)
was calculated using the formula (IR) = 100% - SR. The
IC50of each cell line was calculated.
Flow cytometry analysis
After detachment from the culture dish with 0.25% trypsin,
the cells were washed with phosphate-buffered saline
(PBS) and suspended in PBS at a concentration of 1×106
per mL. The cells were then stained with CD44-FITC and
CD24-PE (BD Bioscience) or with CD133-PE (Ebios-
cience) alone. After incubation on ice in the dark for 30
min, the cells were washed twice with PBS and resuspen-
ded in 1 mL PBS. The cells were kept on ice until flow
cytometry analysis.
Quantitative real-time RT-PCR and RT-PCR
Total RNA was extracted from gemcitabine-resistant cells
and parental cells using Trizol reagent (Invitrogen, Carls-
bad, CA, USA) according to the manufacturer’s instruc-
tions and was reverse transcribed into cDNA using the Pro-
mega AMV reverse transcription system (Promega, Madis-
on WI, USA). Quantitative RT-PCR was performed with
SYBR Green master mix real-time core reagents on an
ABI 7500 (Applied Biosystems) according to the manu-
facturer’s instructions. Primers for quantitative PCR were
as follows:ABCB1
TGATTGCATTTGGAGGACAA-3’, ABCB1 anti-sense:
5’-CCAGAAGGCCAGAGCATAAG-3’. ABCG2 sense:
5’-AGATGGGTTTCCAAGCGTTCAT-3’, ABCG2 anti-
sense, 5’-CCAGTCCCAGTACGACTGTGACA-3’. Shh
sense: 5'-GTGTACTACGAGTCCAAGGCAC-3',
anti-sense: 5'-AGGAAGTCGCTGTAGAGCAGC-3'. Ptc
sense: 5’-TCCCAAGCAAATGTACGAGCA-3’. Ptc Anti-
sense: 5’-TGAGTGGAGTTCTGTGCGACAC-3’. SMO
sense: 5’-CTGGTACGAGGACGTGGAGG-3’, SMO anti-
sense:5’-AGGGTGAAGAGCGTGCAGAG-3’.
sense: 5’-CTCCCGAAGGACAGGTATGTAAC-3’, Gli-1
anti-sense:
CCCTACTCTTTAGGCACTAGAGTTG-3’.
sense: 5' -AGAAAATCTGGCACCACACC-3'. β-actin
anti-sense: 5' -TAGCACAGCCTGGATAGCAA-3'. The
expression of mRNA was normalised to that of the refer-
ence gene, β-actin. Relative quantification of mRNA with-
in the samples was examined using the 2–ΔΔCtmethod. The
primers for SMO used for SW1990 were designed by Kar-
hadkar.(20)
sense:5’-
Shh
Gli-1
5’-
β-actin
Western blot
The concentration of total protein extracted from parental
and gemcitabine-resistant cells was determined with a
BCA Protein Assay Kit (Pierce, USA). Equal amounts of
protein were separated by 10% SDS-PAGE and electro-
phoretically transferred to PVDF membranes (Millipore,
Bedford, USA) using a mini trans-blot (Bio-Rad labor-
atories, Hercules, CA, USA). Rat anti-human SMO,
Gli-1(Millipore, Bedford, USA), ABCB1, and ABCG2
(Abcam, MA, USA) were used to detect the expression
of homologous proteins. β-actin (Santa Cruz Biotechno-
logy, Inc., Santa Cruz, CA, USA) was used as an internal
control. Electrochemiluminescence was performed with a
Chemilmager 5500 imaging system (Alpha Innotech Co.,
San Leandro, CA, USA), according to the manufacturer’s
instructions.
Cyclopamine treatment
CFPAC-1/res and SW1990/res cells were seeded in 6-well
plates (2×105cells per well) for 12 hrs, and then
gemcitabine-free culture media containing 2 μM or 5 μM
cyclopamine were added. After 12 h, 24 h or 48 h, the cells
were harvested. The expression of SMO, Gli1, ABCG2
and ABCB1 before and after cyclopamine treatment was
examined using RT-PCR and western blotting. DMSO
(vehicle) was used as a negative control.
To detect CSC marker changes, gemcitabine-resistant cells
were treated with 2 μM or 5 μM cyclopamine in
gemcitabine-free culture media for 7 days, then the cells
were collected and labelled with flow cytometry antibodies
as described above. The cells cultured in cyclopamine-free,
gemcitabine-free medium were used as a control group.
Cell death and apoptosis assays
After treatment with cyclopamine (2 μM and 5.0 μM) for
24 h, CFPAC-1/res and SW1990/res cells were exposed to
gemcitabine-containing culture media at final gemcitabine
concentrations of 50 μM and 100 μM respectively. After
24 hrs, the cells were harvested and stained with Annexin-
V and PI using the Vybrant Apoptosis Assay Kit (Molecu-
lar Probes) per the manufacturer’s protocol. Briefly, all
cells were harvested by trypsinisation and washed twice
with cold PBS. The pellets were resuspended in 100 μL
1× Annexin binding buffer and 5 μl fluorescein isothiocy-
anate (FITC)–Annexin-V (component A). A 1-μL working
solution of PI at 100 μg/mL was added to each 100 μL of
cell suspension. The cells were incubated on ice for 1 hr,
washed again with cold PBS and re-suspended in 300 μL
1×Annexin-binding buffer. The stained cells were immedi-
ately analysed by flow cytometry.
Results
In vitro establishment of gemcitabine-resistant
pancreatic cancer cells CFPAC-1/res and SW1990/res
After more than 5 months of repetitive exposure to gem-
citabine, both cell lines demonstrated a permanent acquired
chemoresistance. CFPAC-1/res and SW1990/res cells were
able to propagate and be passaged in 50 μM and 100 μM
gemcitabine-containing culture medium respectively. As
detected by the MTT assay, the IC50of CFPAC-1/res and
SW1990/res cells were 68.3 ± 4.5 μM and 306.8 ± 12.3
μM respectively (table 1). Also, the stability of the resistant
phenotype in the absence of gemcitabine was examined in
both CFPAC-1/res and SW1990/res cells. When the cells
were passaged in gemcitabine-free media for 2 weeks, the
IC50remained stable.
CFPAC-1/res and SW1990/res highly express CD44,
CD133 and ABC transporters
We carried out FACS analysis for CD44 and CD24 or
CD133 alone to detect the phenotypic difference between
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the gemcitabine-resistant cells and their parental cells. The
CD44+subfractionwas
gemcitabine-resistant cells compared with their parental
cells (CFPAC-1, 5.64 ± 0.82% vs. 85.07 ± 2.59%;
SW1990, 29.45 ± 1.99% vs. 93.16 ± 2.46%). CD133 was
also found to be highly expressed in both CFPAC-1/res
and SW1990/res cells (CFPAC-1, 28.94 ± 0.5% vs. 75.4 ±
2.25%; SW1990, 59.37 ± 1.69% vs. 95.88 ± 1.47%).
dramaticallyincreased in
Figure 3
Effects of cyclopamine on the expression of SMO, Gli-1,
ABCB1 and ABCG2 in gemcitabine-resistant pancreatic cell
lines. A, Relative mRNA level changes were evaluated using
quantitative RT-PCR, and the expression of β-actin was used as a
control to normalise the relative expression of each gene before
and after cyclopamine treatment. Fold change was analysed using
the 2–ΔΔCtmethod. Values are expressed as means ± SD. B,
Changes in protein level before and after cyclopamine treatment
were tracked by western blot assay. Treatment with 2 μM (+) and 5
μM (++) cyclopamine down-regulated protein levels of SMO, Gli-1,
ABCB1 and ABCG2 in gemcitabine-resistant pancreatic cancer cell
lines, compared with DMSO group (-). Membranes were reprobed
with a β-actin antibody to verify equal loading and transfer. The
molecular size markers (in kDa) are indicated on the right.
Figure 4
Cyclopamine down-regulates CSC markers in gemcitabine-
resistant pancreatic cell lines. Parental CFPAC-1 and SW1990
cells and their gemcitabine-resistant counterparts were double-
labelled with CD44-FITC and CD24-PE or CD133-PE alone. The
percentages of CD44+ and CD133+ cells increased in gemcitabine-
resistant cells (b), compared to parental cells (a). After treatment
with 2 μM cyclopamine for 3 (c) and 7 (d) days, the percentage of
CD44+ and CD133+ cells decreased to close to parental levels.
ABC transporter family member expression is also charac-
teristic of CSCs, which are responsible for the side pop-
ulation (SP) subfraction [21]. We measured the mRNA
expression levels of ABCG2 and ABCB1 in gemcitabine-
resistant cells and their parental cells. The relative ABCG2
mRNA expression in CFPAC-1/res and SW1990/res cells
were increased 3.94 ± 0.65 and 4.44 ± 1.25 fold when com-
pared to their parental cells. The relative ABCB1 mRNA
expression levels in CFPAC-1/res and SW1990/res cells in-
creased by 249.65 ± 220.66- and 166.5 ± 78.24-fold, re-
spectively (fig. 1). The results of the western blot analysis
were consistent with the mRNA expression.
CFPAC-1/res and SW1990/res highly express
Hedgehog signalling members
Since gemcitabine-resistant cells highly expressed some of
the markers of pancreatic cancer stem cells, we hypothes-
ised that they might also highly express some cancer stem
cell-related signalling pathways, such as the HH signalling
pathway, that have been reported to be highly expressed in
CD44+CD24+ESA+ pancreatic cancer stem cells. To test
this hypothesis, the mRNA expression levels of Shh, ptch,
SMO, and Gli-1 were compared in gemcitabine-resistant
cells and parental cells by quantitative real-time PCR, and
the protein levels of SMO and Gli-1 were detected by west-
ern blot. The relative expression levels of Shh mRNA were
increased 2.01 ± 0.74- and 5.19 ± 3.54-fold in CFPAC-1/
res and SW1990/res cells, respectively. The mRNA levels
of Ptch were found to be 1.73 ± 0.59- and 2.79 ± 0.53-fold
increased in CFPAC-1/res and SW1990/res cells respect-
ively (fig. 2a). In CFPAC-1 cells, SMO increased 5.68 ±
0.83-fold when compared to parental cells. Interestingly,
we did not detect any mRNA expression of SMO in par-
ental SW1990 cells, although it can definitely be detected
in SW1990/GZ cells (fig. 2b). The expression of Gli-1 in-
creased 5.75 ± 2.88- and 4.35 ± 1.90-fold in CFPAC-1/res
and SW1990/res cells respectively.
Inhibition of Hedgehog signalling pathway by
cyclopamine inhibits mRNA and protein levels of
SMO, Gli-1, ABCG2 and ABCB1
Because cyclopamine had been shown to inhibit HH sig-
nalling by interfering with the activation of SMO [22, 23],
we treated CFPAC-1/res and SW1990/res with two differ-
ent concentrations (2 μM and 5 μM) of cyclopamine. To
determine the effect of cyclopamine on the expression of
SMO, Gli-1, ABCG2 and ABCB1 in gemcitabine-resistant
cells, the mRNA and protein expression levels before and
after cyclopamine treatment were compared using quantit-
ative RT-PCR and western blotting respectively. Our data
showed that treatment with cyclopamine results in de-
creases in SMO, Gli-1, ABCB1 and ABCG2 in both
gemcitabine-resistant cell lines. Similar results were ob-
tained for protein levels. Western blot analysis showed that
the protein level of SMO was markedly down-regulated by
5 μM cyclopamine at 24–48 hrs after administration of cyc-
lopamine (fig. 3b).
Cyclopamine treatment down-regulates CSC markers
After treatment of gemcitabine-resistant cells with 2 μM or
5 μM cyclopamine for 7 days in gemcitabine-free culture
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medium, the cells cultured in 5 μM cyclopamine underwent
significant cell death and apoptosis, so that the number of
cells became too small for further experiment. The cells
cultured in 2 μM cyclopamine were used to follow the
change in CSC markers. Our data show that the percentage
of CD44+ cells in CFPAC-1/res decreased from 85.31 ±
2.59% to 57.81 ± 1.99% after 3 days and to 17.1 ± 0.55%
after 7 days. The percentage in SW1990/res cells decreased
from 93.16 ± 2.46% to 74.05 ± 1.52% after 3 days and to
36.68 ± 2.44% after 7 days. The percentage of CD133+
cells decreased from 75.4 ± 2.24% to 55.20 ± 1.77% after
3 days and to 31.38 ± 1.99% after 7 days in CFPAC-1/
res cells and from 95.87 ± 1.47% to 76.52 ± 2.97% after
3 days and to 62.76 ± 1.28% after 7 days in SW1990/
res cells. However, after culture in gemcitabine-free and
cyclopamine-free medium, the cells in the control group
displayed no significant differences in the percentages of
CD44+ and CD133+ cells.
Cyclopamine restores gemcitabine sensitivity in
gemcitabine-resistant cells
After treatment with 2 μM cyclopamine for 24 h, signi-
ficantly decreased expression of SMO, Gli-1, ABCG2 and
ABCB1 was detected. We then re-exposed gemcitabine-
resistant cells to gemcitabine-containing cyclopamine-free
culture medium for another 24 h. As shown in figure 4, pre-
treatment with 2 μM or 5 μM cyclopamine significantly re-
duced cell survival in gemcitabine-containing culture me-
dium. For SW1990/res, 53.68 ± 5.24% and 69.99 ± 3.16%
cells underwent cell death when treated with 2 μM and
5 μM cyclopamine respectively, when combined with 100
μM gemcitabine. For CFPAC-1/res cells, 63.60 ± 7.26%
and 76.96 ± 4.28% cells were killed in 2 μM and 5 μM cyc-
lopamine, respectively, when combined with 50 μM gem-
citabine.
Discussion
Chemoresistance is a major cause of gemcitabine treatment
failure in pancreatic adenocarcinoma. After consecutive
treatments, the majority of patients with gemcitabine-
treated pancreatic adenocarcinoma become resistant and
fail to derive benefit from chemotherapy. Hence it is ex-
tremely important to understand the mechanism behind
chemoresistance and to identify predictive markers of in-
herent and acquired chemoresistance to gemcitabine to im-
prove the treatment of these patients.
The cancer stem cell hypothesis provides a new insight into
chemoresistance. Standard chemotherapy can greatly re-
duce tumour bulk but may be less effective on cancer stem
cells. In previous studies [6, 7], tumour stem cells were
found to be enriched in established pancreatic gemcitabine-
resistant cells. After exposure to high-dose gemcitabine,
most of the repopulated cells are CD44+, and they recon-
stituted the resistant cell population. We also carried out
FACS analysis for CD24 and ESA in resistant cells, which
were reported as putative markers of CSCs in pancreat-
ic cancer. However, the percentage of CD24+ and ESA+
cells showed no significant difference. RNA interference of
CD44 inhibited the clonogenic activity of gemcitabine-res-
istant cells [7]. CD133 is also one of the important cancer
stem cell markers [10, 24–26]. In hepatic cancer, CD133+
cancer stem cells confer chemoresistance by preferential
expression of the Akt/PKB survival pathway [27]. In the
present study we found that the CD133+ subfraction in
SW1990/res and CFPAC-1/res was also increased signific-
antly when compared to their parental cells.
When cultured in gemcitabine-free media, a significant de-
crease in CD44 and CD133 expression in gemcitabine-res-
istant cells was observed after 1 week of treatment with
2 μM cyclopamine. At this concentration of cyclopamine,
resistant cells can propagate and be passaged without in-
creased cell death and apoptosis. In Mueller’s research
[11], treatment with cyclopamine combined with rapamy-
cin can abolish CD133+ and CD44+ cell populations in
both primary pancreatic cells and the L3.6pl cell line. Our
data suggest that blockage of the HH signalling pathway
by cyclopamine can also inhibit CSCs markers in
gemcitabine-resistant cells.
Sustained HH signalling activity was detected in pancreatic
adenocarcinoma cell lines isolated from both primary and
metastatic tumours. Inhibition of the HH signalling path-
way by cyclopamine in Cfpac-1 cells can reduce cell pro-
liferation and survival through down-regulation of Gli-1
activity [13]. In our study we evaluated the different
mRNA and protein levels of HH signalling molecules
between the gemcitabine-resistant pancreatic cancer cells
and their parental cell lines. We found upregulation of
Shh, SMO and Gli-1 in gemcitabine-resistant cells. Inter-
estingly, SMO mRNA and protein expression could not
be detected in the parental SW1990 cell line, which is
consistent with Gao’s study [28]. Cyclopamine can block
pancreatic cancer cell growth when SMO mRNA expres-
sion is below the level of detection by QRT-PCR [15]. We
therefore suggest that SMO might be present in parental
SW1990 cells, but at levels too low to be detected by RT-
Figure 5
Cyclopamine restored gemcitabine sensitivity in gemcitabine-
resistant cells as assessed by annexin V and PI staining.
CFPAC-1/res and SW1990/res cells were treated with gemcitabine
(mock control) (c), or DMSO (vector control) (v), or treated with 2
μM cyclopamine for 48 h (1) or 5 μM cyclopamine for 48 h (2),
pretreated by 2 μM cyclopamine for 24 h and then given
gemcitabine-containing culture medium for another 24 h (3), or
given 5 μM cyclopamine pretreatment and then gemcitabine
treatment (4). The concentration of gemcitabine was 50 μM for
CFPAC-1/res and 100 μM for SW1990/res. The cells were then
harvested and stained using a Vybrant Apoptosis Assay Kit, as
described in the materials and methods section. Values are
expressed as means ±SD. *P <0.05.
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