Gefitinib inhibits the proliferation of pancreatic cancer cells via cell cycle arrest.
ABSTRACT High expression of the epidermal growth factor receptor (EGFR) has been implicated in the development of pancreatic cancer. Gefitinib is an orally active and selective EGFR-TKI (EGFR-tyrosine kinase inhibitor) that blocks signal transduction pathways responsible for the proliferation and survival of cancer cells, and other host-dependent processes promoting cancer growth. This study investigated the anticancer effect of gefitinib on human pancreatic cancer cells and the molecular mechanism involved. We first evaluated the effect of gefitinib on cell proliferation with MTT assay and the results demonstrated that gefitinib significantly inhibited the proliferation of pancreatic cancer cells. Flow cytometric analysis showed that gefitinib induced a delay in cell cycle progression and a G0/G1 arrest together with a G2/M block; these were associated with increased expression of p27(Kip1) cyclin-dependent kinase inhibitor combined with decreased expression of aurora B. Besides, luciferase reporter assay revealed that transcriptional mechanism was responsible for the down-regulation of aurora B protein by gefitinib. Overall, the results suggest a mechanistic connection among these events to provide new insights into the mechanism underlying the antiproliferative effect of gefitinib on pancreatic cancer and supplement a theory basis of gefitinib in clinical treatment of pancreatic cancer.
- [Show abstract] [Hide abstract]
ABSTRACT: The multikinase inhibitors sunitinib, sorafenib and axitinib do not only have an impact on tumor growth and angiogenesis, but also on the activity and function of immune effector cells. In this study a comparative analysis of the growth inhibitory properties and apoptosis induction potentials of tyrosine kinase inhibitors on T cells was performed. TKI treatment resulted in a dramatic decrease in T cell proliferation along with distinct impacts on the cell cycle progression. This was at least partially associated with an enhanced induction of apoptosis, although triggered by distinct apoptotic mechanisms. In contrast to sunitinib and sorafenib, axitinib did not affect the mitochondrial membrane potential (Δψm), but resulted in an induction or stabilization of the induced myeloid leukemia cell differentiation protein (Mcl 1) leading to an irreversible arrest in the G2/M cell cycle phase and delayed apoptosis. Furthermore, the sorafenib-mediated suppression of immune effector cells, in particular the reduction of the CD8+ T cell subset along with the down-regulation of key immune cell markers such as CCR7, CD26, CD69, CD25 and CXCR3 was not observed in axitinib-treated immune effector cells. Therefore, rather axitinib than sorafenib seems to be suitable for implementation in complex treatment regimens of cancer patients including immunotherapy.Journal of Biological Chemistry 04/2013; · 4.60 Impact Factor
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ABSTRACT: Drug-resistance to gemcitabine chemotherapy in pancreatic cancer is still an unsolved problem. Combinations of other chemotherapy drugs with gemcitabine have been shown to increase the efficacy of gemcitabine-based treatment. In this study, the effect of berbamine on the antitumor activity of gemcitabine was evaluated in human pancreatic cancer cell lines Bxpc-3 and Panc-1, and the underlying mechanisms were explored. Our results demonstrated that berbamine exhibited a time- and dose-dependent inhibitory effect in the pancreatic cancer cell lines. Berbamine enhanced gemcitabine-induced cell growth inhibition and apoptosis in these cells. Combined treatment of berbamine and gemcitabine resulted in down-regulation of anti-apoptotic proteins (Bcl-2, Bcl-xL) and up-regulation of pro-apoptotic proteins (Bax, Bid). More importantly, berbamine treatment in combination with gemcitabine activated the transforming growth factor-β/Smad (TGF-β/Smad) signaling pathway, as a result of a decrease in Smad7 and an increase in transforming growth factor-β receptor II (TβRII) expression. Changes in downstream targets of Smad7, such as up-regulation of p21 and down-regulation of c-Myc and Cyclin D1 were also observed. Therefore, berbamine could enhance the antitumor activity of gemcitabine by inhibiting cell growth and inducing apoptosis, possibly through the regulation of the expression of apoptosis-related proteins and the activation of TGF-β/Smad signaling pathway. Our study indicates that berbamine may be a promising candidate to be used in combination with gemcitabine for pancreatic cancer treatment. Anat Rec, 2014. © 2014 Wiley Periodicals, Inc.The Anatomical Record Advances in Integrative Anatomy and Evolutionary Biology 03/2014; · 1.34 Impact Factor
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ABSTRACT: Epidermal growth factor receptor (EGFR) is one of the most promising targets for non-small-cell lung cancer (NSCLC). Icotinib, a highly selective EGFR tyrosine kinase inhibitor (EGFR-TKI), has shown promising clinical efficacy and safety in patients with NSCLC. The exact molecular mechanism of icotinib remains unclear. In this study, we first investigated the antiproliferative effect of icotinib on NSCLC cells. Icotinib significantly inhibited proliferation of the EGFR-mutated lung cancer HCC827 cells. The IC50 values at 48 and 72 h were 0.67 and 0.07 μ M, respectively. Flow cytometric analysis showed that icotinib caused the G1 phase arrest and increased the rate of apoptosis in HCC827 cells. The levels of cyclin D1 and cyclin A2 were decreased. The apoptotic process was associated with activation of caspase-3, -8, and poly(ADP-ribose) polymerase (PARP). Further study revealed that icotinib inhibited phosphorylation of EGFR, Akt, and extracellular signal-regulated kinase. In addition, icotinib upregulated ubiquitin ligase Cbl-b expression. These observations suggest that icotinib-induced upregulation of Cbl-b is responsible, at least in part, for the antitumor effect of icotinib via the inhibition of phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways in EGFR-mutated NSCLC cells.BioMed research international. 01/2013; 2013:726375.
THE ANATOMICAL RECORD 292:1122–1127 (2009)
Gefitinib Inhibits the Proliferation
of Pancreatic Cancer Cells via
Cell Cycle Arrest
XIAOHUA ZHOU,†MAQING ZHENG,†FANG CHEN, YUNXIA ZHU, WEI YONG,
HAIYAN LIN, YUJIE SUN, AND XIAO HAN*
Department of Biochemistry and Molecular Biology, Key Laboratory of Human Functional
Genomics of Jiangsu Province, Nanjing Medical University,
Nanjing, People’s Republic of China
High expression of the epidermal growth factor receptor (EGFR) has
been implicated in the development of pancreatic cancer. Gefitinib is an
orally active and selective EGFR-TKI (EGFR-tyrosine kinase inhibitor)
that blocks signal transduction pathways responsible for the proliferation
and survival of cancer cells, and other host-dependent processes promot-
ing cancer growth. This study investigated the anticancer effect of gefiti-
nib on human pancreatic cancer cells and the molecular mechanism
involved. We first evaluated the effect of gefitinib on cell proliferation
with MTT assay and the results demonstrated that gefitinib significantly
inhibited the proliferation of pancreatic cancer cells. Flow cytometric
analysis showed that gefitinib induced a delay in cell cycle progression
and a G0/G1 arrest together with a G2/M block; these were associated
with increased expression of p27Kip1cyclin-dependent kinase inhibitor
combined with decreased expression of aurora B. Besides, luciferase
reporter assay revealed that transcriptional mechanism was responsible
for the down-regulation of aurora B protein by gefitinib. Overall, the
results suggest a mechanistic connection among these events to provide
new insights into the mechanism underlying the antiproliferative effect
of gefitinib on pancreatic cancer and supplement a theory basis of gefiti-
nib in clinical treatment of pancreatic cancer. Anat
C 2009 Wiley-Liss, Inc.
Keywords: gefitinib; pancreatic cancer; cell cycle; aurora B;
Pancreatic cancer is a disease with very poor progno-
sis because conventional chemotherapy only modestly
improves both response and survival rates (Kindler,
2005). As a consequence, improving our understanding
of the molecular events involved in the genesis and pro-
gression of pancreatic cancer has become urgent, also
with a view to identifying potential biomolecular targets
for new therapeutic strategies. Multiple factors have
recently been shown to be possible causes of pancreatic
cancer genesis and aggressiveness, such as high expres-
sion of the epidermal growth factor receptor (EGFR) and
its ligands EGF or TGF-a (detected in more than 90% of
human pancreatic cancer cases) and a role as potential
target of anticancer therapies has been suggested for
EGFR. EGFR is an important receptor involved in sig-
naling pathways implicated in the proliferation and sur-
vival of cancer cells. Dysregulation of EGFR expression
and function occurs in a wide variety of human solid
cancers, including pancreatic cancer (Korc, 1998).
Province; Grant number: BK2003003.
*Correspondence to: Xiao Han, Ph.D., Key Laboratory of
Human Functional Genomics of Jiangsu Province, Nanjing
Medical University, 140 Hanzhong Road, Nanjing 210029, P.R.
China. Fax: þ86-258-686-2731. E-mail: email@example.com
yXiaohua Zhou and Maqing Zheng contributed equally to this
Received 8 March 2009; Accepted 12 May 2009
Published online in Wiley InterScience (www.interscience.wiley.
C 2009 WILEY-LISS, INC.
Gefitinib, an inhibitor of epidermal growth factor re-
ceptor tyrosine kinase (EGFR-TK), is developed for
potential treatment of cancers which overexpress EGFR.
The orally available and reversibly acting drug is highly
specific for EGFR-TK, exhibiting almost no activity
against other TKs and several serine/threonine kinases
(Woodburn et al., 1998; Ciardiello and Tortora, 2001). It
has been well established that gefitinib can suppress the
growth of a range of human cancers expressing high
levels of EGFR, such as lung adenocarcinoma, prostate
cancer, and colon cancer. Moreover, the molecular mech-
anism of gefitinib-mediated inhibition of cell prolif-
eration was mainly dependent on cell cycle arrest (Di
Gennaro et al., 2003; Chang et al., 2004; Sgambato
et al., 2004; Shintani et al., 2004; Koyama et al., 2007).
Gefitinib has also been reported to exert a strong effect
on inhibition of pancreatic cancer cell proliferation (Li
et al., 2004); however, the exact molecular mechanism
involved remains poorly understood.
Therefore, this study was designed to determine
whether gefitinib can inhibit the proliferation of pancre-
atic cancer cells via cell cycle arrest, and if so, to investi-
gate the molecular mechanism involved.
MATERIALS AND METHODS
Dulbecco’s modified Eagle’s medium (DMEM), sodium
pyruvate were from Gibco-BRL (Rockville, MD). New-
born calf serum (NCS) was from PAA Laboratories
(GmbH, Linz, Austria). Gefitinib was purchased from
Sigma Aldrich (St Louis, MO). Anti-aurora B and anti-
p27Kip1rabbit polyclonal antibody were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Horserad-
ish peroxidase-conjugated anti-mouse or anti-rabbit IgG
were obtained from Amersham Pharmacia Biotech (Pis-
cataway, NJ). The Detergent Compatible (DC) Protein
Assay kit was purchased from Bio-Rad Laboratories
(Hercules, CA). Prestained protein markers and restric-
tion enzymes (KpnI and XhoI) were obtained from New
England Biolabs (Beverly, MA). The Luciferase Assay
System was purchased from Promega (Madison, WI).
PANC-1 and CFPAC-1 cells, human pancreatic cancer
cell lines, were obtained from ATCC (Manassas, VA).
The cells were grown in DMEM medium supplemented
with 10% NCS, 10 mmol/L HEPES, 2 mmol/L L-gluta-
mine, 1 mmol/L pyruvate sodium, 100 U/mL penicillin,
and 100 lg/mL streptomycin at 37?C in a humidified
atmosphere containing 95% air and 5% CO2. Cultured
cells were treated with gefitinib (dissolved in DMSO) in
complete DMEM medium. As a control, cultured cells
were incubated in complete DMEM medium containing
DMSO at a final concentration of less than 0.2%.
Cell viability was determined using MTT [3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays.
Briefly, the cells were seeded in 96-well dishes at 1 ?
104cells per well, and treated without (Control) or with
different concentrations of gefitinib for 48 hr. Then each
well was supplemented with 10 lL MTT (Sigma Aldrich)
and incubated for 4 hr at 37?C. The medium was then
removed, and 150 lL DMSO (Sigma Aldrich) was added
to solubilize the MTT formazan. The optical density was
read at 490 nm.
Flow Cytometric Analysis
Cells (1 ? 105cells per well) were cultured in six-well
dishes and treated without (Control) or with 10, 20,
40 lmol/L gefitinib for 48 hr. The cells of each well were
then harvested and fixed with 1 mL 75% ice-cold ethanol
at ?20?C overnight. After fixation, the cells were washed
in PBS and stained with 500 lL propidium iodide solu-
tion (50 lg/mL in PBS) containing 25 lg/mL RNase. The
cells were incubated at room temperature for 0.5 hr in
the dark, and analyzed using a FACSCalibur flow cyto-
meter and Cellquest Pro software (Becton Dickinson
Immunocytometry Systems, San Jose, CA) for data
acquisition and analysis.
Luciferase reporter construct containing aurora B pro-
moter was prepared by using the pGL3-basic vector
(Promega, Madison, WI). The DNA fragment (777 bp)
containing human aurora B promoter was amplified by
PCR from human genomic DNA using the pyrobest PCR
kit (Takara, Otsu, Shiga). The following primers (includ-
ing the sites of restriction enzymes) were synthesized:
AG-30; reverse: 50-TATCTCGAGCGGGTCCAAGGCACTG
CTAC-30. The PCR products were subcloned into the
KpnI and XhoI sites of the pGL3-basic vector to con-
struct pGL3-basic-aurora B-promoter recombinant plas-
mid and then confirmed by sequencing.
Transient Transfection and Luciferase
PANC-1 cells using the pGL3-basic-aurora B-promoter
recombinant plasmid. We used a plasmid containing the
b-galactosidase gene driven by the cytomegalovirus pro-
moter (Clontech Laboratories, Palo Alto, CA) as an inter-
nal control. The PANC-1 cells grown in 24-well dishes
aurora B-promoter and b-galactosidase) using the Lipo-
fectamine Plus transfection kit, according to the manu-
facturer’s instructions. Twelve hours after transfection,
the cells were treated with or without gefitinib for 24 hr,
and then washed with PBS and lysed using 1? passive
lysis buffer. Luciferase activity was measured with a
luminometer (TD-20/20; Turner Designs, CA) using the
Luciferase Assay System. b-galactosidase activity was
detected to normalize any variations in the transfection
Btranscriptionactivity wasassessed in
Western Blot Analysis
PANC-1 cells were lysed in an ice-cold lysis buffer
containing 50 mmol/L Tris-HCl (pH 7.4), 1% NP-40,
150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L phenyl-
methylsulfonyl fluoride (PMSF), and complete proteinase
inhibitor mixture (one tablet per 10 mL; Roche Molecu-
lar Biochemicals, Indianapolis, IN). After protein content
GEFITINIB INHIBITS PANCREATIC CANCER GROWTH
determination using a DC Protein Assay kit, western
blot was performed as described (Han et al., 2002).
Statistical analysis was performed with statistical
analysis software SPSS 11.0 software. Comparisons were
performed using Student’s t test between two groups.
Results are presented as means ? SEM. P < 0.05 was
considered to have significant difference.
Gefitinib Inhibits the Proliferation of Human
Pancreatic Cancer Cells
We first investigated the effect of gefitinib on the pro-
liferation of human pancreatic cancer cells using MTT
assay. Exponentially growing cultures were exposed to
various concentrations of gefitinib for 48 hr. As shown in
Fig. 1, gefitinib inhibited the growth of human pancre-
atic cancer cells in a dose-dependent manner. After 2
days incubation, 20 and 40 lmol/L gefitinib caused a
dramatic growth inhibition in PANC-1 and CFPAC-1
cells. However, there was no change detectable in 2.5, 5,
and 10 lmol/L gefitinib-treated groups.
Gefitinib Inhibits Cell Cycle Progression in
Human Pancreatic Cancer Cells
Because gefitinib could inhibit the proliferation of
human pancreatic cancer cells, propidium iodide stain-
ing and flow cytometric analysis were then performed to
further determine whether the antiproliferative effect of
gefitinib was due to cell cycle arrest. The distribution of
cells in different phases of the cell cycle was determined
after 48 hr in both treated and parallel untreated cul-
tures. As depicted in Fig. 2, after treatment with gefiti-
nib for 48 hr, the percentages of total cells in the G0/G1
phase and G2/M phase strikingly increased while the
percentage of total cells in S phase decreased in a dose-
dependent manner. No subdiploid peak indicative of apo-
ptosis was detected in response to gefitinib treatment.
These results suggested that treatment of gefitinib
induced a G0/G1 arrest and a G2/M block in human pan-
creatic cancer cells.
Up-Regulation of p27Kip1CDK Inhibitor Is
Involved in Gefitinib-Induced G0/G1 Arrest
Cyclin-dependent kinase inhibitor p27Kip1has been
established to be a pivotal regulator involved in G0/G1
arrest induced by gefitinib in many kinds of cancer cells.
To further elucidate the mechanism responsible for G0/
G1 arrest induced by gefitinib in human pancreatic can-
cer cells, we examined PANC-1 cells for this protein.
Western blot analysis (Fig. 3) demonstrated increased
expression of p27Kip1in gefitinib-treated cells and this
accumulation of p27Kip1may be required for gefitinib-
induced G0/G1 arrest.
Decrease of Aurora B Contributes to G2/M
Arrest Induced by Gefitinib
Prior studies have demonstrated that aurora B is an
important mitotic regulator in G2/M phase and deregu-
lation of the expression of aurora B has been implicated
in tumorigenesis. To study the molecular basis of G2/M
arrest caused by gefitinib, we examined the protein
expression of aurora B during cell cycle progression
using western blot analysis under various concentrations
of gefitinib. Figure 4A showed that the protein expres-
sion of aurora B was inhibited by gefitinib after 48 hr
treatment. Quantification data for the western blot re-
vealed that the expression of aurora B protein decreased
to 70.66%, 48.71%, 28.92% versus control group, with
10, 20, 40 lmol/L gefitinib treatment, respectively (data
not shown). To clarify whether the regulation of aurora
B promoter activity was responsible for the down-regula-
tion of its protein expression, we examined the effect of
gefitinib on the promoter activity in the aurora B pro-
moter luciferase fusion plasmid by transient transfection
and luciferase reporter assay. As shown in Fig. 4B,
aurora B luciferase activity decreased in a dose-depend-
ent fashion. In fact, after 24 hr treatment with 20 and
Fig. 1. Effect of gefitinib on cell viability of human pancreatic cancer cells. PANC-1 (A) and CFPAC-1
(B) cells were treated without (Control) or with various concentrations (2.5, 5, 10, 20, 40 lmol/L) of gefiti-
nib for 48 hr. MTT assays were performed to evaluate the cell viability. The data shown are the means ?
SEM of three separate experiments. **P < 0.01 when compared with control group.
ZHOU ET AL.
40 lmol/L gefitinib, the promoter activity of aurora B
decreased to 48.71% (P < 0.05) and 13.23% (P < 0.05),
respectively, when compared with control group. There-
fore, transcriptional mechanism may be responsible for
the down-regulation of aurora B protein by gefitinib.
This study was an attempt to explore the molecular
mechanism of growth arrest induced by gefitinib in
human pancreatic cancer cells. In the initial feasibility
study, we evaluated the effect of gefitinib supplementa-
tion on the growth of pancreatic cancer cells and
detected significant inhibition of the proliferation in a
dose-dependent manner. It has been reported that gefiti-
nib can produce anticancer effect through inducing pro-
grammed cell death (Tracy et al., 2004). However, no
subdiploid peak indicative of apoptosis was detected in
our study, which might be due to compensatory activa-
tion of either downstream pathway effectors or alterna-
tive survival pathway (Camp et al., 2005). Moreover, we
found that gefitinib did cause a delay in cell cycle pro-
gression and a G0/G1 arrest as well as a G2/M block.
These results are in agreement with previous report
demonstrating that the antiproliferative effect of gefiti-
nib was mainly cytostatic and was associated with a
block in G0/G1 phase of the cell cycle (Sgambato et al.,
Our further study to identify the molecular mechanism
responsible for the observed effects indicated that gefiti-
nib produced marked up-regulation of CDK inhibitor
p27Kip1protein which contributed to G0/G1 arrest and
therefore the inhibition of cell proliferation. The results
are consistent with previous evidence demonstrating that
Fig. 2. Flow cytometric analysis of gefitinib-induced cell cycle
arrest. After treatment without (Control) or with various concentrations
of gefitinib, cells were harvested and fixed with 1 mL of 75% ice-cold
ethanol at ?20?C overnight. The next morning, cells were stained with
propidium iodide and analyzed by flow cytometry. The figures are the
results of a typical experiment of PANC-1 (A) and CFPAC-1 (E). Similar
results were obtained in replicate experiments. The percentages of
total cells in the G0/G1 phase (PANC-1, B; CFPAC-1, F), S phase
(PANC-1, C; CFPAC-1, G) and G2/M phase (PANC-1, D; CFPAC-1, H)
are shown as the means ? SEM of three separate experiments. *P <
0.05, **P < 0.01 when compared with control group.
Fig. 3. Western blot analysis of p27Kip1 protein expression. Expo-
nentially growing cultures of PANC-1 cells were treated without (Con-
trol) or with various concentrations of gefitinib. Cells were collected
and total protein extracts were prepared at 48 hr thereafter and
assayed for p27Kip1 (upper part) and b-Actin (lower part, as a loa-
GEFITINIB INHIBITS PANCREATIC CANCER GROWTH
EGF-induced stimulation of growth is mainly associated
with down-regulation of p27Kip1(Ye et al., 1999). More-
over, other groups have tested the molecular mechanism
of the antiproliferative effects of gefitinib on many kinds
of cancer cells. All of their results showed that gefitinib
inhibited cancer cell growth through cell cycle arrest
which was mainly due to the up-regulation of p27Kip1
expression (Chang et al., 2004; Sgambato et al., 2004;
Shintani et al., 2004). Similar findings were also noted by
Budillon (Budillon et al., 2000) and they suggested that
p27Kip1played a key role in gefitinib-induced cell cycle
perturbation by decreasing CDK2 activity and leading to
G0/G1 arrest. Besides, in agreement with the results of
this study, previous observations suggested that blocking
the activation of the EGFR-dependent signal transduction
pathway with anti-EGFR monoclonal antibody induced a
G0/G1 arrest which was associated with an increased
expression of p27Kip1, a reduced CDK2-associated kinase
activity, and no changes in other cell cycle-regulated pro-
teins, including cyclin D1 (Wu et al., 1996).
As a key regulator of G2/M phase of the cell cycle, au-
rora B has been paid plenty of attention in regulation of
cell proliferation. Because defect in mitosis leads to
genomic instability, deregulation of the expression of au-
rora B has been implicated in tumorigenesis. Prior study
indicated that aurora B overexpressed in lots of primary
human cancers (Katayama et al., 1999; Kurai et al.,
2005; Sorrentino et al., 2005; Chieffi et al., 2006; Zeng et
al., 2007) and was a critical target of many anticancer
molecules (Keen and Taylor, 2004). Our study revealed
that gefitinib down-regulated aurora B protein in a dose-
dependent manner, which was consistent with the G2/M
block caused by gefitinib. To further clarify whether the
regulation of aurora B promoter activity was responsible
for the down-regulation of its protein expression, we
examined the effect of gefitinib on the promoter activity
in the aurora B promoter luciferase fusion plasmid by
luciferase reporter assay. Significant inhibition of aurora
B promoter activity by gefitinib was detected. Therefore,
transcriptional mechanism may be responsible for the
down-regulation of aurora B protein by gefitinib.
In conclusion, gefitinib markedly inhibited the prolif-
eration of human pancreatic cancer cells. This inhibition
was caused by a G0/G1 arrest together with a G2/M
block, which was attributable to p27Kip1up-regulation
combined with down-regulation of aurora B. This study
provides new insights into the mechanism underlying
the antiproliferative effect of gefitinib on pancreatic can-
cer and supplements a theory basis of gefitinib in clinical
treatment of pancreatic cancer.
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Fig. 4. Effect of gefitinib on aurora B protein expression and tran-
scriptional activity. (A) PANC-1 cells were treated without (Control) or
with gefitinib (10, 20, 40 lmol/L) for 48 hr and the cell lysates were
obtained for western blot analysis to determine aurora B (upper part)
and b-Actin (lower part, as a loadingcontrol); (B) PANC-1 cells were
transiently transfected with the firefly luciferase reporter construct
pGL3-basic-aurora B-promoter and b-galactosidase (b-gal) plasmid
(as an internal control). Twelve hours after transfection, the cells were
treated without (Control) or with the indicated concentrations of gefiti-
nib (10, 20, 40 lmol/L). Then all the transfected cells were incubated
for an additional 24 h and harvested for luciferase reporter assays.
The relative values of aurora B luciferase activity to b-galactosidase
are shown as the means ? SEM of three independent experiments. *P
< 0.05 when compared with control group.
ZHOU ET AL.