Carcinogenesis vol.26 no.11 pp.
1956–1964, 2005
doi:10.1093/carcin/bgi157
Advance Access publication June 15, 2005
Lupeol, a fruit and vegetable based triterpene, induces apoptotic death of human
pancreatic adenocarcinoma cells via inhibition of Ras signaling pathway
Mohammad Saleemy, Satwinderjeet Kaury,
Mee-Hyang Kweony, Vaqar Mustafa Adhami,
Farrukh Afaq and Hasan Mukhtar

Department of Dermatology, University of Wisconsin, Madison, WI, USA


To whom correspondence should be addressed at Department of
Dermatology, University of Wisconsin, 1300 University Avenue, Medical
Sciences Center, B-25, Madison, WI, 53706, USA. Tel: þ1 608 263 3927,
Fax: þ1 608 263 5223.
Email: hmukhtar@wisc.edu
Pancreatic cancer is an exceptionally aggressive disease,
the treatment of which has largely been unsuccessful due
to higher resistance offered by pancreatic cancer cells to
conventional approaches such as surgery, radiation and/or
chemotherapy. The aberration of Ras oncoprotein has been
linked to the induction of multiple signaling pathways and
to the resistance offered by pancreatic cancer cells to apop-
tosis. Therefore, there is a need for development of new and
effective chemotherapeutic agents which can target mul-
tiple pathways to induce responsiveness of pancreatic can-
cer cells to death signals. In this study, human pancreatic
adenocarcinoma cells AsPC-1 were used to investigate the
effect of Lupeol on cell growth and its effects on the modu-
lation of multiple Ras-induced signaling pathways. Lupeol
caused a dose-dependent inhibition of cell growth as
assessed by MTT assay and induction of apoptosis as
assessed by flow cytometry, fluorescence microscopy and
western blotting. Lupeol treatment to cells was found to
significantly reduce the expression of Ras oncoprotein and
modulate the protein expression of various signaling
molecules involved in PKCa/ODC, PI3K/Akt and
MAPKs pathways along with a significant reduction in the
activation of NFkB signaling pathway. Our data suggest
that Lupeol can adopt a multi-prong strategy to target
multiple signaling pathways leading to induction of apop-
tosis and inhibition of growth of pancreatic cancer cells.
Lupeol could be a potential agent against pancreatic can-
cer, however, further in-depth in vivo studies are
warranted.
Introduction
Pancreatic cancer is the most fatal of all cancers and the fifth
most common cause of cancer-related deaths among both men
and women in the western countries. It is estimated that 32 180
cases of pancreatic cancer will be diagnosed in the United
Abbreviations: DMSO, dimethyl sulfoxide; MAPKs, mitogen-activated
protein kinases; NFkB, nuclear factor kappa B; ODC, ornithine
decarboxylase; PARP, poly(ADP-ribose)polymerase; PI, propidium iodide;
PI3K, phosphatidylinositol 3-kinase; PKCa, protein kinase Ca; PMSF,
phenylmethylsulfonyl fluoride; TdT, terminal deoxynucleotidyl transferase.
y
These three authors contributed equally to this work.
Carcinogenesis vol.26 no.11 # Oxford University Press 2005; all rights reserved.
States in 2005 and almost the same number of pancreatic
cancer-related deaths are projected (1). Since the mortality
from pancreatic cancer compares strikingly with its incidence,
it has become a significant public health concern (1). Long-
term survival of patients with organ-confined disease is only
15%, and in the majority of cases with invasive metastasis
survival is only 4%. Despite these disappointing statistics and
the increase in incidence of pancreatic cancer over the past
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several decades, the molecular and biochemical determinants
of the disease remain poorly understood and no effective
therapeutic regimens to significantly ameliorate the clinical
course or prognosis of this disease is apparent (1).
Thus, pancreatic cancer presents a clinical and experimental
challenge because of its relative resistance to conventional
modes of therapies such as chemotherapy and radiation and
only 4% of such patients are reported to survive after surgery
(1). It is therefore necessary to intensify our efforts for a better
understanding of this disease and for the development of novel
approaches for its prevention and treatment. In more than
90% of pancreatic cancers, the ras oncogene has been shown
to be mutated [(2,3) and references therein]. This mutation is
considered an early genetic event in the development of
pancreatic cancer and results in constitutive activation of an
intracellular pathway leading to cellular proliferation. Several
ras-induced signaling pathways, such as the phosphatidylino-
sitol 3-kinase (PI3K)/Akt (protein kinase B pathway) mitogen-
activated protein kinases (MAPKs) and nuclear factor kappa B
(NFkB) pathways, have been linked to chemo- resistance of
the pancreatic carcinoma cells (3–6). These findings suggest
that Ras oncoprotein could be an important target for devel-
oping agents against pancreatic cancer.
Epidemiological studies have found a lower risk of pancre-
atic cancer in populations with higher consumption of fresh
fruits, vitamin C and dietary fiber (7,8). In addition, consump-
tion of white wine has been reported to have an inverse relation
with the risk of pancreatic cancer (9). These data suggest that
some dietary substances could be effective in reducing pan-
creatic cancer incidence. We argued that those agents, which
have the ability to intervene at more than one critical pathway
in pancreatic carcinogenic process, will have greater advant-
age over other single-target agents. Lupeol, a triterpene
(Figure 1A) is found in various edible plants such as olive,
fig, mango, strawberry, red grapes and medicinal plants used
by native people in North America, Japan, China, Latin
America and Caribbean islands (10–12). Lupeol has been
shown to exhibit strong anti-inflammatory, anti-arthritic,
anti-mutagenic and anti-malarial activity in in vitro and
in vivo systems (10). Lupeol has been shown to act as a potent
inhibitor of protein kinases and serine proteases and of DNA
topoisomerase II, a target for anticancer chemotherapy
(13,14). Lupeol has been reported to induce differentiation
and inhibit the growth of melanoma and leukemia cells
(15–17). Recently, we have shown that Lupeol inhibits
tumor promotion in two-stage mouse skin carcinogenesis by
1956

(B)
100
(A)
80
Cell viability(%)
60
40
20
Lupeol
0
C V
Fig. 1. (A) Structure of Lupeol [Lup-20(29)-en-3b-ol], a triterpene is found in fruits and vegetables. (B) Effect of Lupeol on viability of AsPC-1 cells: The cells
were treated with specified concentrations of Lupeol for 24, 48 and 72 h, and cell viability was determined by MTT assay. The values are represented as the percent
viable cells where vehicle-treated cells were regarded as 100% viable. Data represent mean value of percent viable cells  SE of three independent experiments.
The details are described under Materials and methods.
modulating various signaling pathways (18). In the present
study, employing human pancreatic adenocarcinoma cell line
AsPC-1, we show that Lupeol is a potent multi-target antican-
cer agent that induces apoptotic cell death of pancreatic cancer
cells via modulation of Ras-induced protein kinase Ca
(PKCa)/ornithine decarboxylase (ODC), PI3K/Akt, MAPKs
and NFkB signaling pathways. We suggest that Lupeol may be
an effective agent that should be tested for its in vivo efficacy
against pancreatic cancer.
Materials and methods
Cell culture and reagents
Human pancreatic adenocarcinoma cells AsPC-1 were a gift from Dr Nihal
Ahmad, Department of Dermatology, University of Wisconsin-Madison. The
cells were cultured in RPMI 1640 (ATCC, Manassas VA) in a humidified
atmosphere of 95% air and 5% CO2 at 37 C. Antibodies were procured from
BD Transduction laboratories (San Jose, CA), Santacruz Biotechnology (Santa
Cruz, CA) and Upstate (Charlottesville, VA). Lupeol was purchased from
Sigma Chemical Company (St Louis, MO).
Treatment of cells
Lupeol stock (30 mM) was prepared by dissolving Lupeol in warm alcohol and
diluting in dimethyl sulfoxide (DMSO) in a 1:1 ratio. Working solutions were
prepared by diluting the stock solution with DMSO to get desired final con-
centrations. For dose-dependent studies, the cells (50% confluent) were treated
with Lupeol (30, 40 and 50 mM) for 48 h in complete cell medium. Cells that
served as controls were incubated with the vehicle (alcohol þ DMSO) alone.
The final concentration of DMSO and alcohol was 0.25 and 0.075%,
respectively, in all treatment protocols.
Cell viability assay
The effect of Lupeol on the viability of AsPC-1 cells was determined by
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide) assay.
The cells were plated at 1  104 cells per well in 200 ml of complete culture
medium and treated with 10, 20, 30, 40, 50, 75, 100, 125 mM concentrations of
Lupeol in 96-well microtiter plates. Lupeol stock solutions prepared at 30 mM
concentration were mixed with fresh medium to achieve the desired final
concentration. Each concentration of Lupeol was repeated in 10 wells. After
incubation for 24, 48 and 72 h at 37 C in a humidified incubator, cell viability
Lupeol targets multiple signaling pathways
24 h Treatment
48 h Treatment
72 h Treatment
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10 20 30 40 50 75 100 125
Lupeol (µM)
was determined. MTT [5 mg/ml in phosphate-buffered saline (PBS)] was
added to each well and incubated for 2 h after which the plate was centrifuged
at 1800 r.p.m. (500 g) for 5 min at 4 C. The supernatant was removed from
the wells by aspiration. After careful removal of the medium, 0.1 ml of
buffered DMSO was added to each well, and plates were shaken. The absorb-
ance was recorded on a microplate reader at the wavelength of 540 nm. The
effect of Lupeol on growth inhibition was assessed as percent cell viability
where vehicle-treated cells were taken as 100% viable.
Detection of apoptosis and necrosis by fluorescence microscopy
The Annexin-V-FLUOS apoptosis detection kit (Roche Applied Science,
Indianapolis, IN) was used for the detection of apoptotic and necrotic cells.
This kit uses a dual-staining protocol in which the cells are stained with
annexin and propidium iodide (PI). Cell populations that potentially may be
detected are as follows: viable cells will be non-fluorescent; cells in the
metabolically active stages of apoptosis will stain with annexin (green fluor-
escence) but not with PI (red fluorescence) and necrotic cells with PI staining.
In addition, cells undergoing late stage apoptosis bind both annexin and PI.
Briefly, AsPC-1 cells were grown to 50% confluence on slides and then
treated with Lupeol (30, 40, 50 mM) for 48 h. Apoptosis and necrosis was
detected by the using a Zeiss Axiovert 100 microscope. Briefly, the samples
were excited at 330–380 nm, and the image was observed and photographed
under a combination of a 400 nm dichoric mirror and then 420 nm high-pass
filter.
Quantification of apoptosis
Apoptosis was quantified by terminal deoxynucleotidyl transferase-mediated
dUTP-biotin nick and labeling (TUNEL) method. Briefly, the cells were grown
at a density of 1  106 cells in 100-mm culture dishes and then treated with
Lupeol (30, 40 and 50 mM) for 48 h. The cells were trypsinized, washed with
PBS and processed for labeling with fluorescein-tagged deoxyuridine triphos-
phate nucleotide and PI by use of an APO-DIRECT apoptosis kit obtained
from Phoenix Flow Systems (San Diego, CA) and following the manufac-
turer’s protocol. The labeled cells were then analyzed by flow cytometry.
Total cell lysate and nuclear lysate preparation
The total cell lysate was prepared in cold buffer [50 mM Tris–HCl, 150 mM
NaCl, 1 mM EDTA, 20 mM NaF, 100 mM Na3VO4, 0.5% NP-40, 1% Triton
X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.4] with freshly
added protease inhibitor cocktail (Protease inhibitor Cocktail Set III; Calbio-
chem, La Jolla, CA) in ice. For the preparation of nuclear fractions, the cells
were washed with cold PBS and suspended in 0.4 ml of lysis buffer (10 mM
HEPES, pH 7.9, 10 mM KCI, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT,
1957
M.Saleem et al.
0.5 mM PMSF, Protease Inhibitor Cocktail Set III) in a microfuge tube. The
cells were incubated on ice for 15 min, after which 12.5 ml of 10% Nonidet
P-40 was added and the contents were mixed on a vortex and then centrifuged
for 1 min at 4 C at 14 000 g. The nuclear pellet was resuspended in 25 ml of
ice-cold nuclear extraction buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM
EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, Protease Inhibitor Cocktail
Set III) and incubated on ice for 30 min with intermittent mixing. The tube was
centrifuged for 5 min at 14 000 g at 4 C and the supernatant (nuclear extract)
was stored at 80 C. The protein concentration was determined by BCA assay
(Pierce, Rockford, IL) as per the manufacturer’s protocol.
Western blot analysis
For western blot analysis, 40 mg of the protein was resolved over 8–12%
polyacrylamide gels and transferred to a nitrocellulose membrane. The blot
containing the transferred protein was kept in blocking buffer (5% nonfat
dry milk, 1% Tween-20; in 20 mM TBS, pH 7.6) on a shaker for 1 h at room
temperature followed by incubation with appropriate primary antibody in
blocking buffer for 1 h to overnight at 4 C. This was followed by incubation
with anti-mouse or anti-rabbit secondary antibody conjugated with horse-
radish peroxidase (Amersham Life Sciences) for 1 h. After several washings,
immunoblots were detected by chemiluminescence ECL kit (Amersham Life
Sciences) and autoradiography using XAR-5 film obtained from Eastman
Kodak (Rochester, NY). Immunoblots were scanned by HP Precisionscan
Pro 3.13 (Hewlett-Packard, Palo Alto, CA). Densitometry measurements
of the scanned bands were performed using digitalized scientific software
program UN-SCAN-IT (Silk Scientific Corporation, Orem, UT). Data were
normalized to b-actin and expressed as mean values  SE of three separate sets
of experiments.
Electrophoretic mobility shift assay
To detect NFkB-DNA binding, electrophoretic mobility shift assay (EMSA)
was performed using lightshiftÔ chemiluminiscent kit (Pierce, Rockford, IL)
by following the manufacturer’s protocol. To start with, DNA was biotin
labeled using the biotin 30 end labeling kit (Pierce, Rockford, IL). Briefly, in
a 50 ml reaction buffer, 5 pmol of double-stranded NFkB oligonucleotide 50 -
AGT TGA GGG GAC TTT CCC AGG C-30 ; 30 -TCA ACT CCC CTG AAA
GGG TCC G-50 , 10 ml of 5 TdT (terminal deoxynucleotidyl transferase)
buffer, 5 ml of 5 mM biotin-N4-CTP, 10 U of diluted TdT, and 25 ml of
ultrapure water were incubated at 37 C for 30 min. For supershift assay, the
reaction mixture was incubated for an additional 20 min with or without
antibody for NFkB/p65 (1 ml, Geneka Biotechnology). The reaction was
stopped with 2.5 ml of 0.2 M EDTA. To extract labeled DNA, 50 ml of
chloroform:isoamyl alcohol (24:1) was added to each tube and centrifuged
briefly at 13 000 g. The top aqueous phase containing the labeled DNA was
removed and saved for binding reactions. Each binding reaction contained 1-
binding buffer (100 mM Tris, 500 mM KCl, 10 mM DTT, pH 7.5), 2.5%
glycerol, 5 mM MgCl2, 50 ng/ml poly(dI–dC), 0.05% NP-40, 5 mg of nuclear
extract and 20–50 fmol of biotin-end labeled target DNA. The content was
incubated at room temperature for 20 min. To this reaction mixture, 5 ml of
5 loading buffer was added, subjected to gel electrophoresis on a native
polyacrylamide gel and transferred to a nylon membrane. When the transfer
was complete, DNA was cross-linked to the membrane at 120 mJ/cm2 using a
UV cross-linker equipped with 254 nm bulbs. The biotin end-labeled DNA was
detected using streptavidin–horseradish peroxidase conjugate and a chemilu-
minescent substrate. The membrane was exposed to X-ray film (XAR-5
Amersham Life Science, Arlington Height, IL) and developed using a Kodak
film processor.
Results
Lupeol induces growth inhibition and apoptosis in AsPC-1
cells
We first evaluated the effect of Lupeol on cell viability by
MTT assay. As shown in Figure 1B, treatment of AsPC-1 cells
with Lupeol (10–125 mM) resulted in a dose-dependent inhibi-
tion of cell growth when assessed at 24, 48 or 72 h post-
treatment (Figure 1B). The IC50 value of Lupeol on growth
inhibition was estimated to be 35 mM at 48 h. Based on
these observations, we selected a dose of 30, 40 and 50 mM
and a time period of 48 h post-Lupeol treatment for further
mechanistic studies.
We next determined whether Lupeol-mediated loss of cell
viability in AsPC-1 cells is a result of induction of apoptosis.
The induction of apoptosis by Lupeol (30–50 mM) was evident
1958
from the morphology of cells, as assessed by fluorescence
microscopy after labeling the cells with annexin and PI
(Figure 2, upper panel). As shown by representative pictures
(Figure 2, upper panel), Lupeol treatment resulted in induction
of apoptosis in a dose-dependent manner. These data also show
that Lupeol treatment also resulted in necrosis of these cells,
which may be a secondary event in the apoptotic process. We
next quantified the extent of apoptosis by flow-cytometric
analysis of Lupeol-treated cells labeled with bromo-
deoxyuridine triphosphate and PI. As shown by the data in
Figure 2 (bottom panel), compared with control, Lupeol treat-
ment resulted in 0.3, 8.6 and 22.0% TUNEL-positive cells
at 30, 40 and 50 mM at 48 h post-treatment, respectively.
Consistent with the fluorescence microscopy data, TUNEL
assay by flow cytometry revealed that treatment of AsPC-1
cells with Lupeol resulted in a dose-dependent induction of
apoptosis.
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Lupeol induces PARP cleavage and increases Bax protein
expression in AsPC-1 cells
Since poly(ADP-ribosylation) is a post-translational modifica-
tion of proteins which plays a crucial role in DNA repair and
cell death, poly(ADP-ribose)polymerase (PARP), a DNA nick
sensor, serves as one of the best-known biomarkers of apop-
tosis (19). During apoptosis, PARP protein is cleaved into
an 85 kDa C-terminal fragment, with a reduced catalytic activ-
ity, and a 24 kDa N-terminal peptide, which retains the DNA
binding domains. Next, we assessed the effect of Lupeol
(30–50 mM) treatment of cells on PARP cleavage. The
PARP cleavage analysis showed that the full-size PARP
(116 kDa) protein was cleaved to yield an 85 kDa fragment
after treatment of cells with Lupeol at 30, 40 and 50 mM
concentrations at 48 h post-treatment (Figure 3). The expres-
sion of Bax (pro-apoptotic protein) and Bcl-2 (anti-apoptotic
protein) have been reported to play a crucial role in apoptotic
response mediated by many agents (20,21). We next determ-
ined the effect of Lupeol treatment of cells on the protein
levels of Bax and Bcl-2. The immunoblot analysis exhibited
a significant increase in the protein expression of Bax in
Lupeol-treated cells (Figure 3). However, the protein expres-
sion of Bcl-2 did not exhibit any change by Lupeol treatment at
any dose.
Lupeol induces activation of caspases in AsPC-1 cells
Caspases receive the upstream death signals and are the final
executioners of apoptosis (22,23). In most cancer cells,
caspases are present in the pro-forms (inactive) and require
cleavage of protein to become active to participate in the
process of apoptosis. Through this process, the pro-forms of
caspases are lost and the expression of their active forms
(cleaved products) increases during apoptosis (22,23). We
next performed immunoblot analysis of the members of cas-
pases in the cell lysates obtained from Lupeol-treated cells. We
have used antibodies that specifically recognize the pro-forms
and cleaved products (active forms) of caspases. As shown in
Figure 4, Lupeol-treated cells did not exhibit any change in the
expression level of procaspase-3, however, the expression
of active caspase-3 was found to be increased in such cells
(Figure 4). Further Lupeol treatment induced the expression of
caspases-8 and -9 in a dose-dependent manner which is evident
from the decrease in the protein expression of their pro-forms
(inactive forms) with a concomitant increase in their active
forms as compared with vehicle-treated control (Figure 4).
 Lupeol targets multiple signaling pathways

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Fig. 2. Lupeol induces apoptosis in AsPC-1 cells as assessed by fluorescence microscopy (upper panel) and by flow cytometry (bottom panel). The upper
panel contains the representative micrographs of AsPC-1 cells undergoing apoptosis induced by treatment with Lupeol as assessed by fluorescence microscopy.
The cells were treated with vehicle alone or specified concentration of Lupeol for 48 h. Apoptosis and necrosis was detected by a Zeiss Axiovert 100 microscope
as described in Materials and methods. The samples were excited at 330–380 nm, and the image was observed and photographed under a combination of a
400 nm dichoric mirror and then 420 nm high pass filter. The bottom panel is the quantitative representation of Lupeol-induced apoptosis in AsPC-1 cells
as assessed by flow cytometry. The cells were treated with Lupeol (30–50 mM for 48 h), labeled with deoxyuridine triphosphate using terminal deoxynucleotide
transferase and PI by using an apoptosis APO-DIRECT kit obtained from Phoenix Flow Systems (San Diego, CA) as per vendor’s protocol followed by
flow cytometry. Cells showing deoxyuridine triphosphate fluorescence above that of control population, as indicated by the line in each histogram, are considered
as apoptotic cells. The images shown here are representative of three independent experiments with similar results. The values shown inside each graph indicate
the percent apoptotic cells. The details are described under Materials and methods.

Lupeol decreases the expression of Ras, PKCa, ODC proteins

Fig. 3. Effect of Lupeol treatment on PARP cleavage and on protein
expression of Bax, Bcl-2 in AsPC-1 cells. The cells were treated with vehicle
(DMSO þ alcohol) only or specified concentrations of Lupeol for 48 h,
harvested and total cell lysates were prepared. PARP cleavage and the
expression of Bax and Bcl-2 were determined by western blot analysis. Equal
loading was confirmed by stripping immunoblots and reprobing them for
b-actin. Values given above each lane on the immunoblot represents the
relative density of the bands normalized to b-actin. The immunoblots shown
here are representative of three independent experiments with similar results.
The details are described under Materials and methods.
and inhibits PI3K/Akt in AsPC-1 cells
Many cancers including pancreatic cancer exhibit aberrant Ras
signaling that leads to chronic up-regulation of the Ras path-
way and accumulation of Ras oncoprotein in transformed
cells (24). PKCa has been implicated in Ras signaling and
transformed cells have been reported to have high levels of
PKCa (25). In various cancer types, Ras-induced PKCa has
been reported to activate ODC, an oncogene which is highly
activated during the cellular proliferation (25–27). Next, we
determined the effect of Lupeol treatment of cells on the
expression of Ras, PKCa and ODC proteins. It is evident
from the data of immunoblot analysis that treatment of Lupeol
significantly reduced the protein expression of Ras in a dose-
dependent manner (Figure 5). Similarly, treatment of Lupeol
to cells also caused a significant reduction in the protein
expression of ODC and PKCa (Figure 5). These findings
suggest that Lupeol exerts a preferential inhibitory effect on
Ras/PKCa/ODC pathway leading to reduction in pancreatic
cancer cell growth.
The frequent aberration in Ras signaling is reported to cause
constitutive activation of PI3K/Akt during the development of
pancreatic cancer in humans [(6,28) and references therein].
We next investigated the effect of Lupeol treatment of cells on
PI3K/Akt signaling pathway. Lupeol treatment of cells was
found to cause a significant reduction in the expression of
regulatory subunit of PI3K (p85) protein (Figure 5). Similarly,
Lupeol caused a significant dose-dependent decrease in the
phosphorylation of Akt protein in cells (Figure 5). The treat-
ment of Lupeol did not cause any change in the protein levels
of total Akt (Figure 5). These results suggest the inhibitory
potential of Lupeol against PI3K/Akt pathway, which is highly
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M.Saleem et al.

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Fig. 4. Effect of Lupeol treatment on protein expression of caspases-3, -8 and
-9 in AsPC-1 cells. The cells were treated with vehicle (DMSO þ alcohol)
only or specified concentrations of Lupeol for 48 h, harvested and total cell
lysates were prepared. The expression of proforms and cleaved products of
caspase -3, -8 and -9 were determined by western blot analysis. Equal
loading was confirmed by stripping immunoblots and reprobing them for
b-actin. Values given above each lane on the immunoblot represents the
relative density of the bands normalized to b-actin. The immunoblots shown
here are representative of three independent experiments with similar results.
The details are described under Materials and methods. Fig. 5. Effect of Lupeol treatment on the expression of Ras, PKCa, ODC,
PI3K (P85), pAkt and total Akt, phospho-p38 and Erk1/2 proteins in AsPC-1
cells: the cells were treated with vehicle (DMSO þ alcohol) only or specified
concentrations of Lupeol for 48 h, harvested and total cell lysates were
activated during the development and progression of pancre- prepared. The expression of Ras, PKCa, ODC, PI3K(P85), pAkt and total
atic cancer. Akt, p38 and Erk1/2 proteins total cell lysate was determined by western blot
analysis. Equal loading was confirmed by stripping immunoblots and
reprobing them for b-actin. Values given above each lane on the immunoblot
Lupeol inhibits the expression of MAPK proteins p38 and represents the relative density of the bands normalized to b-actin. The
Erk1/2 in AsPC-1 cells immunoblots shown here are representative of three independent
Constitutive levels of Erk1/2 protein have been reported to be experiments with similar results. The details are described under Materials
very low in pancreatic cancer cells and studies have linked the and methods.
dysregulation of MAPKs with tumor cell survival in various
cancer types including pancreatic adenocarcinoma (3,29,30).
JNK1 protein (data not shown). Taken together, these obser-
Therefore, we determined whether MAPKs contributed to
Lupeol-induced apoptosis in our model. We addressed this vations indicate that activation of Erk1/2 protein and decrease
in expression levels of phospho-p38 protein might be contrib-
question by determining the effect of Lupeol treatment on
uting towards apoptosis of cells induced by Lupeol.
expression levels of Erk1/2, JNK and p38 MAPK proteins.
As shown in Figure 5, exposure of cells to Lupeol resulted in a
dose-dependent activation (phosphorylation) of Erk1/2. Lupeol inhibits phosphorylation of IkBa and NF-kB/p65 in
Lupeol treatment caused a dose-dependent decrease in AsPC-1 cells
the level of phospho-p38 MAPK (Figure 5). Unlike ERK1/2 A number of studies clarified the role of NFkB in tumor cell
and p38, however, Lupeol treatment did not alter the level of survival by inhibiting apoptosis (31). The activation of NFkB
1960
 Lupeol targets multiple signaling pathways

pathway in pancreatic cancer cells has been linked to
their resistance to conventional chemotherapy (32). The
phosphorylation of inhibitory protein IkBa is a critical
step in the pathway of NFkB activation that results in the
translocation of NFkB molecule to nucleus from cytosol,
which ultimately leads to the survival of tumor cells. The
translocation of NFkB to nucleus is preceded by the phos-
phorylation of its p65 subunit and therefore is an important
event. Next, we investigated the effect of Lupeol treatment on
the phosphorylation of IkBa and NFkB/p65 in cells. As shown
by immunoblot analysis (Figure 6A), Lupeol treatment
resulted in a dose-dependent inhibition of phosphoryla-
tion of NFkB/p65 protein in these cells. The inhibition of
phosphorylation of NF-kB/p65 molecule was concomitant
with a decrease in the phosphorylation of IkBa protein
(Figure 6A).
NFkB–DNA binding is reduced in Lupeol-treated AsPC-1
cells
The translocation of NFkB to nucleus is marked by its binding
with DNA to activate the expression of various genes leading
to the tumor cell survival (31). We next performed EMSA to
investigate the effect of Lupeol treatment on NFkB–DNA
binding activity. As shown in Figure 5B, untreated and vehicle
control cells exhibited a constitutively increased NFkB–DNA
binding activity; however, in cells treated with Lupeol, a
significant decrease in NFkB–DNA binding activity was
observed (Figure 6B). The effect of Lupeol on NFkB–DNA
binding in cells was concomitant with the effect of Lupeol on
phosphorylation of NF-kB/p65 subunit (Figure 6A). As is
evident from results of supershift assay using anti-p65 anti-
bodies, Lupeol was observed to specifically decrease the levels
of p65 subunit of NF-kB in the nucleus of cells (Figure 6C).

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Fig. 6. (A) Effect of Lupeol treatment on the phosphorylation of IkBa protein and p65-subunit of NFkB transcriptional factor in AsPC-1 cells. The cells were
treated with vehicle (DMSO þ alcohol) only or specified concentrations of Lupeol for 48 h, harvested and total cell lysates were prepared. Equal loading was
confirmed by stripping immunoblots and reprobing it for b-actin. Values given above each blot represents the relative density of the bands normalized to b-actin.
The immunoblots shown here are representative of three independent experiments with similar results. The details are described under Materials and methods.
(B) Effect of Lupeol treatment on the binding of NFkB transcriptional factor with DNA in AsPC-1 cells as assessed by EMSA. After 48 h of treatment with
Lupeol, the cells were harvested, nuclear lysates were prepared and DNA binding was determined by EMSA as described under Materials and methods.
I, II, and III refer to internal experimental controls, where I represents biotin–EBNA (Epstein–Barr virus nuclear antigen) control DNA, II represents biotin–EBNA
control DNA and EBNA extract, and III represents biotin–EBNA control DNA and EBNA extract plus 200-fold molar excess of EBNA DNA. In control
number I, no protein extract for DNA to bind resulted in an unshifted band. In control number II, sufficient target protein leads to DNA–protein binding resulting in
shift detected by comparison to band at position I. Control number II demonstrated that the signal shift observed could be prevented by competition from
excess unlabelled DNA. The data shown here are representative of three independent experiments with similar results. (C) Effect of Lupeol treatment on the
binding of p65-subunit of NFkB transcriptional factor with DNA in AsPC-1 cells. The cells were treated with vehicle only or specified concentrations of
Lupeol for 48 h, and then processed for nuclear lysate. NFkB/DNA binding activity was determined by Supershift assay using anti-p65 antibody and EMSA kit
from Pierce (Rockford, IL). The details are described under Materials and methods. The data shown here are representative of three independent experiments
with similar results, ns represents non-specific binding.

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M.Saleem et al.
Discussion
The aggressive nature of pancreatic adenocarcinoma is related
to several abnormalities in growth factors and their receptors
that affect the downstream signal transduction pathways
involved in the control of growth and differentiation (4).
These perturbations confer a tremendous survival and growth
advantage to pancreatic cancer cells. The acquisition of an
apoptosis-resistant phenotype by pancreatic cancer cells may
also upset the success of conventional radio- and chemother-
apies (32). In addition to perturbations in cell cycle control,
resistance to apoptosis may promote tumor growth and meta-
stasis. Apoptosis resistance mechanisms seem to vary among
different tumor types and have only begun to be characterized
at the molecular level.
Elucidation of molecular events during pancreatic cancer
development has led to several distinct therapeutic advances
and many novel agents and inhibitors including monoclonal
antibodies have been developed and are undergoing clinical
trials (4,32–34). However, many of these agents are not effect-
ive alone and have to be combined for maximum efficacy
leading to undesirable and sometimes fatal side effects. One
promising approach could be identification of non-nutrient
dietary constituents that can target multiple pathways without
causing any undesirable toxicity. Here, we describe the use-
fulness of Lupeol, a novel non-nutrient dietary agent against
human pancreatic cancer cells. In the current study, we dem-
onstrate that Lupeol treatment induces a dose-dependent death
of human pancreatic cancer cells. From our data, it is evident
that the induction of cell death is mediated through apoptosis.
We also found that Lupeol-induced apoptosis of pancreatic
cells is mediated through the activation of caspases-3, -8 and
-9. The observation that expression levels of procaspase-3 did
not exhibit any significant change upon Lupeol treatment
could be explained through a possible involvement of a feed-
back mechanism, which restores depleted procaspase-3 levels
at intracellular level. The only other pro-apoptotic effect of
lupeol reported so far is on mouse melanoma and human
leukemia cells (15–17).
Transformed cells have been shown to possess a dysregu-
lated apoptotic machinery (35). Several pro-apoptotic proteins
are either lost or diminished and many anti-apoptotic proteins
are activated during the development of cancer (35). As is
evident from our data, induction of pro-apoptotic Bax protein
by Lupeol indicates that Lupeol may rectify the errors in
apoptotic machinery of pancreatic cancer cells. Although
many studies have shown that natural agents decrease the
expression of anti-apoptotic protein Bcl-2 with a concomitant
increase in the expression of pro-apoptotic Bax protein and
shift the balance towards apoptosis, we did not observe
any change in the Bcl-2 protein expression in Lupeol-treated
pancreatic cancer cells. However, with an increase in Bax
expression the shift is indeed towards apoptosis. Since our
data suggest that Lupeol is a multi-target agent, other mech-
anisms of apoptosis could not be ruled out.
Other contributing molecular changes in pancreatic adeno-
carcinoma include activation of oncogenes and inactivation of
tumor suppressor genes (2–5). The ras oncogene is thought to
play an important role in the growth of pancreatic cancer,
because an activated (mutated) ras gene is found in 90% of
human pancreatic cancers (2–5). The ras mutation results in
the accumulation of Ras oncoprotein, and constitutive activa-
tion of various intracellular signaling pathways, leading to
1962
cellular proliferation (2–6). Targeting the ras gene has been
shown to inhibit the growth of metastatic pancreatic cells (4).
In the present study, we observed that treatment of human
pancreatic cancer cells with Lupeol decreases the protein
expression of Ras in a dose-dependent manner.
Ras protein is reported to regulate various downstream
signaling pathways such as the PI3K/Akt pathway. The
growth-promoting potential of the PI3K/Akt pathway and its
anti-apoptotic properties are closely linked to the resistance of
cancer cells to a broad spectrum of apoptotic stimuli and
several studies have demonstrated that treatment with the
PI3K inhibitors substantially enhances apoptosis (4–6,36,37).
Studies have shown that inhibitors of PI3K/Akt induce dose-
dependent apoptosis of pancreatic cancer cells and inhibit
tumor growth of pancreatic cancer xenografts (37,38). In the
current study, we show that treatment of pancreatic cancer
cells with Lupeol reduced the expression of PI3K and Akt
Downloaded from carcin.oxfordjournals.org at William Jewell College on February 26, 2011
proteins in a dose-dependent manner. This is consistent with
our earlier report where we demonstrated that Lupeol inhibited
the PI3K/Akt signaling pathway in vivo during two-stage
mouse skin tumorigenesis (18). These results suggest that
Lupeol has an efficiency to modulate the signaling pathways
which are known to promote pancreatic cancer cell growth
and survival.
The effects mediated by aberrantly activated Ras in human
cancers are likely to be very complex and are currently not
completely understood. Both Ras and PI3K/Akt signaling
pathways have been reported to regulate the expression of
MAPKs during the progression of various cancer types includ-
ing pancreatic cancer (3,4,29,30). Chronic stimulation of the
Ras/Raf/MEK/ERK pathway by oncogenic Ras is thought to
promote cellular transformation and this pathway has been
reported to be activated in various cancer types, however,
Erk1/2 activation does not always correlate with the expres-
sion of mutant ras or other oncogenes (3). Constitutive levels
of Erk1/2 proteins have been reported to be very low in
pancreatic cancer cells despite Ras activation and Erk1/2
inhibition in pancreatic cancer cells have been linked with
the resistance to apoptosis [(3, 30) and references therein].
As evident from our data, the correlation of significant cell
death with increased expression of Erk1/2 proteins in Lupeol-
treated cells is evidence that low levels of Erk1/2 protein
have an association with tumor cell survival in pancreatic
cancer. Our data are supported by recent studies showing that
chemotherapeutic agent-induced apoptosis in pancreatic can-
cer cells is through Erk1/2 activation. It has been demonstrated
that p38 MAPK activation triggers an anti-apoptotic response
to tumor cells, although there have also been some contrasting
reports that p38 MAPK activation (phosphorylation) is central
to the pro-apoptotic effects in some cancer cell lines (39,40).
Recent reports have demonstrated a link between the Ras
oncoprotein and p38 MAPK activation in transformed cells
(41). It is evident from our data that Lupeol treatment signi-
ficantly decreases the expression of phospho-p38 MAPK in
cells leading to their apoptotic death.
Ras oncoprotein has been shown to contribute to constitutive
activation of NFkB signaling in pancreatic cancer through the
activation of PI3K/Akt and MAPK pathways (32). Wang et al.
reported that p65 subunit of NFkB was constitutively activated
in 67% of pancreatic adenocarcinomas and a positive cor-
relation exists between the activation of NFkB and Ras
oncoprotein during the proliferation of pancreatic cancer
cells (42). NFkB plays an important role in tumor resistance

Fig. 7. A schematic representation of the action of Lupeol with respect to various signaling pathways in AsPC-1 cell. " Represents increased expression;
# represents decreased expression; ‘ represents inhibition; and ! represents induction. See online Supplementary material for a color version of this figure.
to apoptosis and studies have shown that pancreatic cancer
cells resistant to apoptosis exhibit a high basal NFkB activity
(31,43). Interestingly, we found that treatment of Lupeol inhib-
ited expression of NFkB and phosphorylation of IkBa protein
in pancreatic cancer cells. Because Lupeol inhibits IkBa phos-
phorylation, our study suggests that the effect of Lupeol on
NFkB/p65 is through inhibition of phosphorylation of IkBa
which in turn is regulated by Ras and PI3K/Akt pathways.
The Ras oncoprotein is also reported to regulate PKCa
(23,24). PKCa has been reported to be involved in the regula-
tion of apoptosis and in animal models, tumorigenicity of
pancreatic cancer cells has been directly related to increased
expression of PKCa protein (25,26). Numerous studies have
shown that PKC levels are high in pancreatic cancer patients
and increased PKC levels offer resistance to apoptosis of
pancreatic cancer cells (32). In this scenario, Lupeol with an
efficacy of targeting PKCa protein seems to be a promising
agent against the growth of pancreatic cancer cells as it exhib-
its an inhibitory potential against PKCa protein and induces
apoptosis of pancreatic cancer cells in a dose-dependent
manner.
Ras-induced PKCa is known to induce the ODC protein,
the first and the rate-limiting enzyme in the biosynthesis of
polyamines expression during the development of various
cancer types (26,27,44). Elevated levels of ODC gene products
are consistently detected in transformed cell lines, virtually
all-animal tumors and in certain tissues predisposed to tumori-
genesis (24,25). Because tumor formation can be prevented by
the agents that block induction of ODC, several ODC inhibit-
ors have been tested against various types of cancers [(18) and
references therein]. These data demonstrate that Lupeol is
a potent inhibitor of ODC expression and is consistent with
our earlier report where we showed that Lupeol inhibits ODC
activity and expression in vivo (18).
Based on the outcome of this study, we suggest Lupeol could
provide a multi-prong beneficial strategy for targeting multiple
signaling pathways leading to apoptosis and inhibition of
Lupeol targets multiple signaling pathways
Downloaded from carcin.oxfordjournals.org at William Jewell College on February 26, 2011
growth of pancreatic cancer cells. The schematic representa-
tion of the action of Lupeol is depicted in Figure 7. This may
be explained by modulation of NFkB mediated by Ras-
induced pathways such as PKCa/ODC, PI3K/Akt and MAPK
signaling. Since pancreatic cancer is resistant to conventional
chemotherapeutic regimens, Lupeol could be a potential agent
to overcome this resistance. Further in-depth in vivo studies are
warranted to verify this suggestion and are presently under
investigation in our laboratory.
Supplementary material
Supplementary material can be found at: http://carcin.
oxfordjournals.org/
Acknowledgements
Author (S.K.) was recipient of BOYSCAST fellowship from Department of
Science and Technology, Government of India. This work was supported by
United States Public Health Service grants RO1 CA 78809 and RO1 CA
101039.
Conflict of Interest Statement: None declared.
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Received April 12, 2005; revised May 26, 2005; accepted June 7, 2005