IJC
International Journal of Cancer
Epicatechin-rich cocoa polyphenol inhibits Kras-activated
pancreatic ductal carcinoma cell growth in vitro and
in a mouse model
Hifzur Rahman Siddique1, D. Joshua Liao2, Shrawan Kumar Mishra1, Todd Schuster3, Lei Wang4, Brock Matter5,
Paul M. Campbell6, Peter Villalta5, Sanjeev Nanda7, Yibin Deng4 and Mohammad Saleem1
1
Department of Molecular Chemoprevention and Therapeutics, The Hormel Institute, University of Minnesota, Austin, MN
Department of Translational Cancer Research, The Hormel Institute, University of Minnesota, Austin, MN
3
Shared Instruments Center, The Hormel Institute, University of Minnesota, Austin, MN
4
Department of Cell Death and Cancer Genetics, The Hormel Institute, University of Minnesota, Austin, MN
5
Mass Spectrometry Facility, Department of Analytical Biochemistry, Masonic Cancer Center, University of Minnesota, Minneapolis, MN
6
Department of Drug Discovery, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL
7
Department of Internal Medicine, Mayo Clinic Health System, Austin, MN
2
Cancer Therapy
Activated Kras gene coupled with activation of Akt and nuclear factor-kappa B (NF-jB) triggers the development of pancreatic
intraepithelial neoplasia, the precursor lesion for pancreatic ductal adenocarcinoma (PDAC) in humans. Therefore, intervention
at premalignant stage of disease is considered as an ideal strategy to delay the tumor development. Pancreatic malignant
tumor cell lines are widely used; however, there are not relevant cell-based models representing premalignant stages of PDAC
to test intervention agents. By employing a novel Kras-driven cell-based model representing premalignant and malignant
stages of PDAC, we investigated the efficacy of ACTICOA-grade cocoa polyphenol (CP) as a potent chemopreventive agent
under in vitro and in vivo conditions. It is noteworthy that several human intervention/clinical trials have successfully
established the pharmacological benefits of cocoa-based foods. The liquid chromatography (LC)–mass spectrometry (MS)/MS
data confirmed epicatechin as the major polyphenol of CP. Normal, nontumorigenic and tumorigenic pancreatic ductal
epithelial (PDE) cells (exhibiting varying Kras activity) were treated with CP and epicatechin. CP and epicatechin treatments
induced no effect on normal PDE cells, however, caused a decrease in the (i) proliferation, (ii) guanosine triphosphate (GTP)bound Ras protein, (iii) Akt phosphorylation and (iv) NF-jB transcriptional activity of premalignant and malignant Krasactivated PDE cells. Further, oral administration of CP (25 mg/kg) inhibited the growth of Kras-PDE cell-originated tumors in a
xenograft mouse model. LC–MS/MS analysis of the blood showed epicatechin to be bioavailable to mice after CP
consumption. We suggest that (i) Kras-driven cell-based model is an excellent model for testing intervention agents and (ii)
CP is a promising chemopreventive agent for inhibiting PDAC development.
Pancreatic ductal adenocarcinoma (PDAC) is one of the lethal cancers found in humans. The severity and lethality of
this type of cancer can be ascertained from the report published by American Cancer Society, which projected 37,660
deaths out of 44,030 new PDAC patients in year 2011 in the
Key words: Kras, preneoplastic, cocoa polyphenol, epicatechin,
pancreatic cancer
Grant sponsor: The Hormel Institute
DOI: 10.1002/ijc.27409
History: Received 22 Oct 2011; Revised 5 Dec 2011; Accepted 9 Dec
2011; Online 20 December 2011
Correspondence to: Mohammad Saleem, Department of Molecular
Chemoprevention and Therapeutics, The Hormel Institute,
University of Minnesota, Austin, MN 55912, USA and Department of
Laboratory Medicine and Pathology, University of Minnesota-Twin
Cities Campus, Minneapolis, MN 55912, USA, Tel.: þ507-437-9662,
Fax: 507-437-9606, E-mail: msbhat@umn.edu
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Int. J. Cancer: 131, 1720–1731 (2012) V
United States.1 Chemotherapy and surgery are the common
treatment options for PDAC, and unfortunately, both the
treatment options have dismal outcome in patients. This is
evident from the reports that PDAC patients have a survival
rate of only 4% after surgery or chemotherapy.1 PDAC putatively evolves through multistage neoplastic transformation
process, which is reflected in a series of histologically welldefined precursor lesions termed as pancreatic intraepithelial
neoplasia (PanIN).1–3 Molecular analysis of PanIN lesions
has revealed progressively accumulating genetic abnormalities
involving several oncogenes.4 Mutations of the Kras gene on
chromosome 12P are one of the earliest genetic abnormalities
observed during PDAC development and found in approximately 36, 44 and 87% of cancer-associated PanIN-1A,
PanIN-1B and PanIN-2/3 lesions, respectively.4 It is reported
that Akt signaling pathway couple with Kras activation to
trigger the PDAC development in humans.5 Activation or
overexpression of Akt is found in about 20–70% of PDAC
cases.5,6 nuclear factor-kappa B (NF-jB) has been well
1721
Siddique et al.
Material and Methods
Chemicals
13
C3-catechin was from Polysciences (Warrington, PA). The
solvent-free cocoa-polyphenols (CP) powder was kindly provided by Barry Callebaut Innovations (Lebbeke, Belgium). It
was isolated from nonroasted cocoa beans using the ACTICOA process developed by Barry Callebaut. Catechin, epicaC 2011 UICC
Int. J. Cancer: 131, 1720–1731 (2012) V
techin and epicatechin gallate were purchased from LKT Laboratories (St. Paul, MN).
CP powder preparation for liquid chromatography
(LC)–Mass spectrometry (MS)/MS
CP powder was mixed with water at a concentration of 0.5
mg/mL. The solution was repeatedly vortexed and sonicated for
20 sec to insure all soluble material entered solution. The 13C3catechin was used an internal standard. An aliquot of the stock
solution was further diluted with 0.1% formic acid and mixed
with 200 pmol of 13C3-catechin to achieve a final CP concentration of 0.01 mg/mL prior to LC–MS/MS analysis. The mass
spectrometer was operated in the electrospray ionization
(ESI)-MS/MS mode, and analysis was performed by selective
reaction monitoring. The first quadruple was set to isolate the
deprotonated molecules (MH]) of catechin (m/z 289.05),
epicatechin (m/z 289.05), 13C3-catechin (m/z 292.05) and epicatechin gallate (m/z 441.08). Fragmentation was induced in the
second quadruple with a collision gas pressure (Ar) of 1.5 and
collision energy of 15 V. The third quadruple was set to detect
product ions at m/z 244.0 (catechin and epicatechin), m/z
248.0 (13C3-catechin) and m/z 289.05 (epicatechin gallate).
Cell line generation and characterization
As described earlier, PDE cells were immortalized by employing the method of Lee et al.9,18 Briefly, primary cell cultures
isolated from pancreatic ducts were sequentially infected with
retroviral vectors to express human telomerase (hTERT) and
the E6 and E7 proteins. From this precursor cell line, a
matched pair was generated with or without expression of
the constitutively activated Kras (G12D) mutant (designated
E6E7 and E6E7-Ras, respectively).9 Finally, these two cell
lines were infected with SV40 (designated E6E7-st and E6E7Kras-st, respectively). The resulting mass populations were
maintained at 5% CO2 in high-glucose dulbecco’s modified
eagle medium (DMEM) (Life Technologies, Carlsbad, CA)
supplemented with 10% fetal bovine serum (FBS).
Cell growth assay
The effect of CP on the growth of cells was determined by
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazoliumbromide
(MTT) assay as described earlier.19 Cells were treated with CP
dissolved in water at concentrations of 0.001–0.1% for 48 hr.
Treatment of cells
For biochemical studies in vitro, we employed tumorigenic
E6E7-Kras-st cells. Cells at 70% confluence were treated with
freshly prepared CP (0.001–0.1% in water) or with water
(control). At 48 hr, cells were harvested and processed for
nuclear- and whole-cell lysate preparation by employing
methods as described previously.19–21
Bromodeoxyuridine labeling
E6E7-Kras-st cells were seeded at 1 105 cells/mL in 24-well
plates. Cells were subsequently grown in serum-free DMEM
Cancer Therapy
documented to play a role in PDAC development.7 Kras has
been reported to drive activation of both NF-jB and Akt signaling.8 Studying molecular changes during premalignant
stages of PDAC has not been fully successful in past due to
the lack of model systems. Recently, Campbell et al. (coauthor of this study) developed a unique cell-based model that
represents the progression stages of premalignant lesion of
PDAC development.9 The uniqueness of this model is that it
is based on Kras/Akt molecular activation within the pancreatic ductal epithelial (PDE) cells thus has high relevance to
human PDAC.9 As PDAC has a poor prognosis at advanced
stage, a modest delay in the progression of premalignant to
malignant carcinoma through chemopreventive intervention
could result in a substantial reduction of incidence of this
disease and more importantly, improve the quality of life of
the population at risk. Premalignant lesions thus offer a
potential substrate for intervention.
Epidemiological studies suggest a reduced risk of PDAC
with high consumption of vegetables and fruits.10,11 Preclinical and clinical studies have established beneficial pharmacological effects of cocoa-based functional foods such as functional chocolate on human health.12–17 This is evident from
the outcome of 30 human intervention trials with dark chocolate and cocoa-based foods (Ref. 13 and references therein).
These intervention trials showed that consumption of cocoabased foods and drinks is associated with short-term
improvements in delayed oxidation of low-density lipoprotein
cholesterol, improved endothelial function, lowered blood
pressure and improved platelet function in human
patients.14–17 An epidemiological study of elderly men
showed that blood pressure was significantly lowered (with a
lower incidence of cardiovascular disease-related death) in
the group consuming the highest dark chocolates.15 Cocoa
beans are a concentrated source of procyanidins, flavan-3-ols,
epicatechin and catechin.13–17 The pharmacological effects of
cocoa are attributed largely to the presence of high-epicatechin content.13 As major polyphenols are lost during classical
extraction, attempts have been made to prepare cocoa-based
functional foods (those are rich in polyphenolic content) by
employing techniques such as ACTICOA. In this study, we
tested our hypothesis that ACTICOA-grade epicatechin-rich
cocoa polyphenols (CP) extract could be a potential chemopreventive agent against premalignant and malignant stage of
PDAC. We provide data showing that epicatechin-rich CP by
targeting the Kras/Akt/NF-jB signaling module inhibits the
growth of human premalignant and malignant Kras-activated
PDE cells in vitro and in vivo.
1722
plus 1% charcoal stripped (CS)-FBS for an initial 24 hr. The
medium was then replaced with either CP or epicatechin
plus the bromodeoxyuridine (BrdU) labeling reagent (1:1,000;
BD Biosciences, Bedford, MA). Cells were incubated with
BrdU labeling reagent for 24 hr. Cells were then washed with
phosphate buffered saline (PBS) and fixed with acid–ethanol
(5:95) for 30 min. Fixed cells were incubated with normal serum for 1 hr followed by 1-hr incubation with mouse monoclonal anti-BrdU antibody (Cell Signaling, Danvers, MA). After washing with PBS, the cells were incubated for 1 hr with
HRP-conjugated sheep antimouse IgG. Labeled nuclei were
counted in 10 different fields (20 magnification) per well in
two separate experiments, and mean values expressed as a
percentage of the total number of cells per field.
pulse. The mixture of supernatant and the agarose pellet was
resolved over a polyacrylamide gel and analyzed for GTPbound Kras protein by employing Western blot technique.
GTPcS and guanosine diphosphate (GDP) were used as positive and negative controls, respectively.
Western blot analysis
This was performed as described earlier.19–23
Electrophoretic mobility shift assay
This was performed as described earlier.22 After every 2 days,
media were changed, and treatment agents (CP and epicatechin) in fresh media were added to colonies. After 21 days in
culture, colonies were counted in five random three-dimensional fields per well.
This was performed by employing lightshiftTM chemiluminiscent kit (Pierce, Rockford, IL) as per manufacturer’s protocol.
After 48 hr of treatment with CP and epicatechin, the cells
were harvested, nuclear lysates were prepared, and electrophoretic mobility shift assay (EMSA) was performed as
described earlier.19–21 The following oligonucleotides were
used for double stranded NF-jB: 50 -AGT TGA GGG GAC
TTT CCC AGG C-30 ; 30 -TCA ACT CCC CTG AAA GGG
TCC G-50 . The biotin end-labeled DNA was detected using
streptavidin–horseradish peroxidase (HRP) conjugate and a
chemiluminescent substrate.
Cell-cycle analysis
NF-jB transcriptional activation assay
Cells (60% confluent) were starved for 12 hr to arrest them
in G0 phase of the cell cycle, after which they were treated
with CP (0.025%) and epicatechin (0.025%) in complete
media for 48 hr. The harvested cells were processed for cellcycle analysis as described earlier.23
The human NF-jB reporter plasmid (pTAL-NF-jB-luc) was
purchased from Clontech (Mountain View, CA). Cells seeded
at a density of 5 104 cells/well were transfected with the
plasmids (1 lg/million cells) for 48 hr. Renilla luciferase (50
ng/million cells, pRL-TK; Promega, Madison, WI) was used
as an internal control. In addition, the same amounts of
empty vectors were transfected in control cells. At 12 hr
post-transfection, fresh media were added with CP (0.01–
0.1%) and epicatechin (0.01–0.05%) and incubated for 48 hr.
Cells were harvested, and transcriptional activity was measured in terms of luciferase activity using dual-luciferase
reporter assay system (Promega, Madison, WI). Relative luciferase activity was calculated with the values from vector
group with or without treated group.
Growth transformation assay
Quantification of apoptosis
Cancer Therapy
Epicatechin-rich cocoa polyphenol inhibition
After incubating with CP (0.025%) and epicatechin (0.025%)
for 48 hr, cells were harvested and washed with PBS (containing 2.5% FBS). Followed by washing with PBS, cells were
incubated for 5 min at room temperature with Annexin
V-fluorescein isothiocyanate (FITC) plus propidium iodide
(PI) and analyzed on a Becton Dickinson fluorescence activated cell sorting (FACS) Calibur flow cytometer (BD Biosciences, San Jose, CA).
Tumorigenicity studies in athymic nude mice
Kras activation assay
Visualization of active Kras protein (guanosine triphosphate
(GTP)-bound Kras) expression was performed using a glutathione S-transferase-bound Raf-Ras-binding pull-down assay
kit (Millipore, Mountain view, CA). Briefly, cells were collected in cold PBS, and lysates were prepared. Ras assay reagent (5–10 lg; Raf-1 ras binding domain (RBD), agarose)
was added to 0.5 mL of cell lysate. The reaction mixture was
incubated for 45 min at 4 C with gentle agitation, and the
agarose beads were pelleted by brief centrifugation (14,000g,
4 C). Supernatant was removed and discarded followed by
washing the beads by adding 0.5 mL lysate buffer. Agarose
beads were resuspended in 40 lL of 2 Laemmli reducing
sample buffer. Beads were pelleted by brief centrifugation followed by addition of 2 lL of 1 M dithiothreitol followed by
boiling for 5 min. Beads were collected by microcentrifuge
Athymic (nu/nu) male nude mice (6 weeks old; Harlan Laboratory) were housed under pathogen-free conditions with a 12
hr light/12 hr dark schedule and fed with an autoclaved diet
ad libitum. For these studies, we employed Kras-activated PDE
(E6E7-Kras-st) cells, because they develop rapid tumors.9 A
total of 1 106 E6E7-Kras-st cells suspended in 50 lL of
media and 50 lL of matrigel (BD Biosciences, Bedford, MA)
were inoculated subcutaneously into the right flank of each
athymic nude mouse (6 weeks old). Implantation of cells into
mice produced visible tumors with a meant latent period of 14
days. Animals were then randomly divided into two groups,
with 10 animals in each group. Animals in Group 1 received
autoclaved water (100 lL) by oral gavage and served as control. Animals in Group 2 received freshly prepared CP (25
mg/kg) in 100 lL of water by oral gavage 3-days/week. The
dose of CP was selected on the basis of published studies.24,25
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1723
Siddique et al.
Body weights were recorded 7 days weekly throughout the
study. Tumor growth was measured weekly, and tumor volumes were calculated as described earlier.21 Before 2 hr of sacrifice, each animal received an intraperitoneal administration
of BrdU labeling reagent (10 mL/kg; Invitrogen, Camarillo,
CA) to label proliferating cells within tumors. From the harvested tissues, lysates were prepared, and paraffin tumor sections were prepared on slides. The lysates were stored at
80 C. All procedures conducted were in accordance with the
guidelines for the use and care of laboratory animals.
respect to the expression of various proteins. A Kaplan–Meier
survival analysis with the corresponding Log-Rank and Linear Regression analysis was used to measure the rate of mean
tumor volume growth as a function of time. A p-value of
<0.05 was considered to be statistically significant.
Results
LC–MS/MS analysis of CP
Quantitative analysis showed that CP contains 6,625 nmol/g
of epicatechin, 376 nmol/g of catechin and 292 nmol/g of
epicatechin gallate (Fig. 1a).
Immunohistochemical analysis
Plasma sample preparation for capillary ultra performance
liquid chromatography (UPLC)-ESI-MS/MS
Whole blood was collected by mandibular bleeding into anticoagulant citrate-treated tubes. Cells are removed from plasma
by centrifugation at 2,000g using a refrigerated centrifuge. The
resulting supernatant was designated plasma. Following centrifugation, plasma samples were transferred into a clean polypropylene tube using a Pasteur pipette and kept at 80 C for
further analysis. Aliquots of plasma (50 lL) were removed and
mixed with 200 pmol of 13C3-catechin, 25 U of sulfatase and
500 U of b-glucuronidase in 100 mM sodium acetate (pH 5).
Samples incubated at 37 C for 45 min were loaded on to prepared Oasis hydrophilic-lipophilic balance (HLB) (30 mg)
solid phase extraction cartridges (Waters, Milford, MA). Samples were washed with 1 mL of water and eluted with 80%
acetonitrile containing 0.1% formic acid. Samples were dried
under vacuum and reconstituted in 20 lL of 0.1% formic acid
followed by centrifugation at 16,000g to separate any insoluble
material. The supernatant was transferred to autosampler vials.
Capillary UPLC-ESI-MS/MS
Quantitative analyses of catechin, epicatechin, epicatechin
gallate and 13C3-catechin in plasma samples were performed
on a triple stage quadrupole (TSQ) Quantum Ultra mass
spectrometer coupled with a Waters NanoAcquity capillary
UPLC. A BEH130 Shield RP18 column (0.3 100 mm, 1.7
lm) was eluted at room temperature at a flow rate of 8 lL/
min with a gradient of acetonitrile (Solvent B) in water (Solvent A), both containing 0.1% formic acid. The solvent composition was changed from 5 to 20% B in 8 min, then
ramped up to 40% B at 11 min and then ramped down to
5% and equilibrated by 20 min. The mass spectrometer was
operated in the ESI-MS/MS mode, and quantitative analysis
was performed by selective reaction monitoring.
CP inhibits the growth of Kras-activated PDE cells
We investigated the effect of CP on the growth of normal
PDE (wild-type Kras), transformed PDE (E6E7-st; SV40transformed/wild-type Kras) and Kras-activated PDE cells
(nontumorigenic E6E7-Kras and tumorigenic E6E7-Kras-st;
mutant Kras). By employing MTT assay, CP (0.001–0.1%)
treatment decreased growth of both nontumorigenic and
tumorigenic Kras-activated PDE cells (E6E7-Kras and E6E7Kras-st), while sparing normal PDE cells (Fig. 1b). The IC50
for CP was estimated to be 0.042% for nontumorigenic
(E6E7-Kras) and 0.028% for tumorigenic (E6E7-Kras-st) cells
(Fig. 1b). As epicatechin is a major constituent of CP, we
investigated the effect of epicatechin on the growth of cells.
The IC50 of epicatechin for Kras-activated nontumorogenic
and tumorigenic PDE cells was estimated to be 0.075 and
0.05%, respectively (Fig. 1c). Based on these observations, we
selected (i) tumorigenic Kras-activated PDE cells (E6E7-Krasst) and (ii) doses ranging from 0.025 to 0.05% (862–1,724
lM) of epicatechin for further biochemical studies.
We investigated the effect of CP and epicatechin treatment
on the rate of proliferation of E6E7-Kras-st cells by employing
BrdU proliferation assay. Both CP and epicatechin treatments
significantly decreased BrdU uptake by cells (Fig. 1d). Next,
we asked whether long-term (21 days) treatment, with CP and
epicatechin could exert greater activity on the formation of
colonies, which allows an investigation over a longer period of
time and which mimics cellular physiology in vivo. The colony-forming ability of cells was observed to be significantly
inhibited by CP and epicatechin treatments (Fig. 1e).
CP induces G1 cell-cycle arrest in E6E7-Kras-st cells
We evaluated whether the inhibition in the growth of E6E7Kras-st cells involves an arrest of cells at specific check
point(s) in the cell cycle. By employing FACS analysis, we
observed that CP and epicatechin treatments caused arrest of
cells at G0–G1 phase of cell cycle. CP and epicatechin treatments increased 14 and 11% of cells in G1 phase, respectively, with a concomitant decrease in the number of cells in
S and G2 phase of the cell cycle (Fig. 2a).
Statistical analyses
CP induces apoptosis in E6E7-Kras-st cells
Student’s t test for independent analysis was applied to evaluate differences between the treated and untreated cells with
We determined whether CP-induced inhibition in the growth
of E6E7-Kras-st cells is a result of induction of apoptosis.
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Cancer Therapy
Immunohistochemical staining was performed as described
earlier.26 Appropriate primary antibodies (anti-BrdU, antipNF-jB and anti-p38; Cell Signaling, Danvers, MA) at a
dilution of 1:50 were used.
Figure 1. LC–MS/MS analysis of cocoa polyphenols (CP) powder and the effect of CP and epicatechin on growth, proliferation and clonogenic
potential in human PDE cells. (a) Chromatogram showing the relative abundance of catechin and epicatechin. 13C3-catechin was used as internal
standard as described under ‘‘Material and Methods’’ section. Inset: structure of catechin and epicatechin is indicated within (a). (b, c) Line graphs
represent the effect of CP and epicatechin on growth of human normal pancreatic PDE (wild Kras), transformed PDE (E6E7-st; wild Kras) and Krasactivated PDE (nontumorigenic E6E7-Kras and tumorigenic E6E7-Kras-st) cells. Cells were treated with specified concentrations of CP and epicatechin
for 48 hr, and cell growth was determined by MTT assay. Each concentration of CP and epicatechin (0.001–0.10%) was repeated in 10 wells. The
values are represented as percent viable cells where vehicle (water)-treated cells were regarded as 100% viable. Data represent mean value of percent
viable cells 6 SE of three independent experiments. The details are described under ‘‘Material and Methods’’ section.
(d) Histogram represents the effect of CP and epicatechin on proliferation of E6E7-Kras-st cells as assessed by BrdU uptake assay. Cells were treated
with 0.025–0.05% CP and epicatechin. The number of BrdU-positive cells per field was determined after 24 hr of continuous labeling. Labeled nuclei
were counted in 10 different high fields (20 magnification) per well in two separate experiments, and mean values are expressed as a percentage of
the total number of cells per field. *Significantly different (p < 0.05) from the control (vehicle treated) group. (e) Histogram represents the number of
colonies formed by E6E7-Kras-st cells treated with CP and epicatechin. Cells seeded in agarose and incubated at 37 C were treated with CP and
epicatechin as described under ‘‘Material and Methods’’ section. After 21 days of incubation, the cells were stained with crystal violet/methanol, and
colonies were counted. Each bar in the histogram represents mean 6 SE. *p < 0.05. All experiments were repeated three times.
1725
Figure 2. Effect of CP and epicatechin on cell cycle and apoptosis in Kras-activated cells. (a) Effect of CP and epicatechin on cell cycle in
E6E7-Kras-st cells. The cells were synchronized in G0 phase by depleting the serum for 12 hr. After 12 hr of serum starvation, cells were
treated with vehicle or CP or epicatechin for 48 hr in complete serum containing medium and were analyzed by flow cytometry. The
percentages of cells in the G0/G1, S and G2/M phases were calculated using cell fit computer software. Other details are described under
‘‘Material and Methods’’ section. The data shown here are from a representative experiment repeated three times with similar results.
(b) Quantitative estimation of CP and epicatechin-induced apoptosis in E6E7-Kras-st cells as assessed by flow cytometry. Cells were treated
with CP and epicatechin for 48 hr, labeled with Annexin-V and PI. Intact cells were gated in the forward scatter (FSC)/side scatter (SSC)
plot to exclude small debris. Cells in the lower right quadrant of the FL1/FL2 dot plot (labeled with Annexin V-FITC only) are considered to
be in early apoptosis, and cells in the upper right quadrant (labeled with Annexin V-FITC and PI) are in late apoptosis/necrosis. The images
shown here are representative of three independent experiments with similar results. (c) Immunoblot images represent effect of CP
treatment on protein levels of native, cleaved PARP, procaspase-3 and 8 and cleaved caspase-3 and 8 as determined by immunoblot
analysis. (d) Immunoblot images represent effect of CP treatment on the protein levels of Bcl-xL, Bcl-2 and Bax in cells. (e) Histogram
shows Bax/Bcl-2 ratio. Cells were treated with vehicle (water) or different concentrations of CP for 48 hr, harvested and total cell lysates
were prepared. For immunoblot analysis, equal loading was confirmed by stripping the membrane and reprobing them for b-actin. The
immunoblots shown here are representative of three independent experiments with similar results. The details are described under
‘‘Material and Methods’’ section.
V stands for vehicle-treated cells.
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Cancer Therapy
Siddique et al.
1726
FACS analysis of cells showed that CP and epicatechin treatment resulted in 34 and 32% of apoptotic cells, respectively
(Fig. 2b). The cleavage of poly (ADP-ribose) polymerase
(PARP) and caspases proteins result in their activation, thus
are considered important surrogate biomarkers of apoptosis.27 CP-treated cells exhibited a reduction in native
PARP116, procaspase-3 and 8 levels (Fig. 2c). However,
cleaved PARP85 and cleaved Caspase-3 and 8 were
observed to be increased by CP treatment (Fig. 2c).
Bcl-xL and Bcl-2 antiapoptotic proteins play an important
role in the survival of premalignant and malignant cells.27
Prior studies have shown that the proapoptotic effects of Bax
(a protein known to antagonize the Bcl-2) is overridden by
antiapoptotic Bcl-2 family proteins.27 We investigated if the
CP-induced apoptosis is driven by modulation of Bcl-xL, Bcl2 and Bax. CP treatment decreased protein levels of Bcl-xL,
Bcl-2, and increased the levels of Bax in E6E7-Kras-st PDE
cells (Fig. 2d). As assessed by the relative densities of the immunoblot bands, CP was found to cause a significant shift in
Bax/Bcl-2 ratio favoring apoptosis of cells (Fig. 2e). Similar
data were observed with epicatechin treatment (data not
shown). These data suggest that CP-induced growth inhibition was a result of apoptosis of Kras-activated PDE cells.
Cancer Therapy
CP decreases the level of active-Kras (GTP-bound)
in E6E7-Kras-st cells
Aberrant Kras signaling in transformed PDE cells is a hallmark of PDAC development.5,26 We determined the effect of
CP treatment of Kras-activated PDE cells on the level of
active form of Ras protein (GTP-bound) by employing an
affinity pull-down assay. CP and epicatechin treatments reduced
the GTP-bound active Ras protein levels in E6E7-Kras-st cells,
however, did not cause any significant effect on total Ras protein (Fig. 3a). CP treatment was observed to have no effect on
Ras protein in normal PDE cells (data not shown).
Ras protein is known to activate mitogen-activated protein
kinase (MAPK) signaling in premalignant and malignant
lesions.27 Constitutive activation of MAPK protein is reported
to contribute to cell proliferation and apoptosis inhibition.28–31
CP treatment caused a dose-dependent decrease in the level of
phospo-p38 MAPK in Kras-activated PDE cells (Fig. 3b). Epicatechin treatment caused a decrease in phospo-P38 levels in cells
(data not shown).
CP inhibits phosphatidylinositol 3-kinases (PI3K)/Akt in
E6E7-Kras-st cells
Aberrant Kras signaling is reported to cause activation of
PI3K/Akt during the development of human PDAC.4–6 We
investigated the effect of CP treatment on the PI3K/Akt signaling in E6E7-Kras-st cells. CP treatment significantly
reduced the levels of regulatory subunit of PI3K (p85) protein and decreased phosphorylation of Akt protein in cells
(Fig. 3b). Similar data were observed with epicatechin treatment (data not shown).
Epicatechin-rich cocoa polyphenol inhibition
CP inhibits phosphorylation of IjBa and NF-jB/p65 in
E6E7-Kras-st cells
Kras is reported to contribute to constitutive activation of
NF-jB signaling during the developmental stages of human
PDAC through the activation of PI3K/Akt and MAPK pathways.20,32 There is considerable evidence that activation of
NF-jB contributes to evasion of apoptosis and malignant
progression during PDAC development.7,32 The phosphorylations of IjBa inhibitory protein and p65 subunit of NF-jB
are critical steps that result in the translocation of NF-jB to
nucleus. We investigated the effect of CP treatment on the
phosphorylation of IjBa and NF-jB/p65 in cells. CP treatment dose dependently inhibited the phosphorylation of NFjB/p65 in cells (Fig. 3c). The inhibition of phosphorylation
of NF-jB/p65 molecule was concomitant to a reduced phosphorylation of IjBa protein (Fig. 3c). Similar data were
observed with epicatechin treatment (data not shown).
NF-jB-DNA binding is reduced in CP-treated
E6E7-Kras-st cells
The translocation of NF-jB to nucleus is marked by its binding with DNA to activate the expression of target genes leading to the cell survival and proliferation.7,33,34 By employing
EMSA, we observed that Kras-activated PDE cells exhibit an
increased NF-jB DNA-binding activity. However, CP and
epicatechin treatments decreased NF-jB-DNA-binding activity in Kras-activated PDE cells (Fig. 3d).
CP inhibits the transcriptional activity of NF-jB
in E6E7-Kras-st cells
NF-jB upon binding to the consensus regions activates the transcription of several cell proliferation-associated genes.7,32–34 By
employing a luciferase-based reporter assay, we observed that CP
and epicatechin treatment inhibited the transcriptional activation
of NF-jB (80–90%) in E6E7-Kras-st cells (Figs. 3e and 3f).
Effect of CP on tumorigenicity of E6E7-Kras-st cells
in an athymic nude mouse model
AS CP was observed to be effective in inhibiting the growth of
E6E7-Kras-st cells in vitro, we next determined whether these
results could be translated under in vivo situations. CP-feeding
did not (i) cause any loss in the body weight, (ii) food intake
and (iii) exhibit any apparent signs of toxicity in animals. The
average volume of tumors in control mice increased as a function of time and reached a preset end point of 1,000 mm3 in
42 days postinoculation. However, at this time, the average tumor volume was only 377 mm3 in mice receiving oral-feeding
of CP (25 mg/kg in 100 lL of water; Fig. 4a). Next, we determined the effect of CP-feeding on latency period of tumors in
animals. The observed differences for tumor growth in CP-fed
mice as compared to control mice were statistically significant
with p < 0.05 (Fig. 4b). Approximately, 50% of mice which
received CP-feeding did not cross the preset end point of the
tumor volume of 1,000 mm3 even at the end of 10th week
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Figure 3. Effect of CP and epicatechin on Kras/Akt/NF-jB signaling in Kras-activated PDE cells. (a) Immunoblot images represent the effect
of CP and epicatechin on activated Ras (GTP-bound Ras), assessed by pull-down assay as described in ‘‘Material and Methods’’ section.
Equal loading was confirmed by stripping the membrane and reprobing them for b-actin and total Ras protein levels. Cell lysates loaded
with GDP and GTPcS were used as negative and positive controls, respectively. (b) Representative immunoblot images show protein levels
of pP38, PI3K (p85), pAkt and total Akt in Kras-activated cells (c) Immunoblot images represent protein levels of pIjBa, IjBa, pNF-jB and
NF-jB in Kras-activated cells. Equal loading was confirmed by stripping the membranes and reprobing them for b-actin. (d) Immunoblot
image represents NF-jB DNA-binding activity in E6E7-Kras-st cells. Nuclear lysates were prepared from cells, and DNA binding was
determined by EMSA as described under ‘‘Material and Methods’’ section. 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 III demonstrated that the observed signal shift could be prevented by
competition from excess unlabeled DNA. Lane number IV represents biotin-NF-jB control DNA without protein extract. The data shown here
are representative of three independent experiments with similar results. ns represents nonspecific binding. (e, f) Histograms represent the
effect of CP and epicatechin treatment on the transcriptional activation of NF-jB in E6E7-Kras-st cells. NF-jB-luc-transfected E6E7-Kras-st
cells were treated with CP and epicatechin, and the transcriptional activity was measured in terms of luciferase activity as described under
‘‘Material and Methods’’ section. Each bar in the histogram represents mean 6 SE. *p < 0.05. All experiments were repeated three times.
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Siddique et al.
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Epicatechin-rich cocoa polyphenol inhibition
Figure 4. Effect of CP-feeding on the tumorigenicity of E6E7-Kras-st cells and expression levels of different proteins under in vivo
conditions. (a) The graphical representation of data shows the effect of CP treatment on growth of tumors from E6E7-Kras-st cells
implanted in athymic nude mice. The growth was measured in terms of average volume of tumors as a function of time. Data are
represented as mean 6 SE (n ¼ 10). *p < 0.05 from the control group (b) The graphical representation of the data depicts the number of
mice remaining with tumor volumes <1000 mm3 after treatment with water alone or CP for indicated weeks (n ¼ 10). (c) Representative
photomicrographs (20 magnification) show immunohistochemical staining for BrdU in tumor sections of water-fed (C1) and CP-fed (C2)
mice. The arrows in the micrographs represent regions exhibiting immunoreactivity. The immunostaining data were confirmed in all
specimens from each group (n ¼ 10). (d) Histogram represents the average tumor weight of the E6E7-Kras-st cell-derived tumors of waterfed and CP-fed animals. Each bar in the histogram represents mean 6 SE. *p < 0.05. (e) Immunoblot images represent the protein levels
of native, cleaved PARP, Bcl-xL, Bcl-2 and Bax. (f) Histogram shows Bax/Bcl-2 ratio measured from densitometry analysis of immunoblots
of Bcl2 and Bax. (g) Representative photomicrographs (20 magnification) show immunohistochemical staining for phospo-p38 (G1–4) and
pNF-jB (G5–8) in tumor sections of water-fed (G1–2 and G5–6) and CP-fed (G3–4 and G7–8) mice. The arrows in the micrographs
represent regions exhibiting immunoreactivity. The immunostaining data were confirmed in all specimens from each group. (h)
Immunoblots represent the effect of CP-feeding on the protein level of phospo-P38, PI3K, pAkt, Akt, phospo-NF-jB and NF-jB. For
immunoblot analysis equal loading was confirmed by stripping the membrane and reprobing them for b-actin.
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Siddique et al.
Figure 5. Evaluation of epicatechin blood levels in mice after CP consumption. (a) The chromatograms represent the detection of standard
(13C3-catechin) and epicatechin as assessed by LC–MS/MS as described under ‘‘Material and Methods’’ section. The peaks in the graphs
represent the retention times at which analytes were detected. (b) The quantitative estimation of catechin and epicatechin levels in plasma
samples collected at different time intervals from mice receiving one-time feeding of CP (100 mg/kg).
CP-feeding inhibits proliferation of tumor cells in vivo
BrdU immunostaining of tumors showed that CP-fed mice
exhibited reduced rate of proliferation of cells within tumors
(Fig. 4c). When compared for average tumor weight, the
observed differences between control and CP-fed group were
statistically significant (Fig. 4d).
CP-feeding induces apoptosis of tumor cells in vivo
We investigated the effect of CP-feeding on apoptotic
markers in tumor tissues harvested from control and CP-fed
mice. CP-feeding was observed to induce the levels of
PARP85 and decrease the level of PARP116 protein in
tumors (Fig. 4e). In addition, Bax/Bcl-2 ratio was found to be
higher in tumor tissues of CP-fed mice suggesting increased
apoptosis of cells within tumor tissues of this group (Fig. 4f).
We next determined the effect of CP-feeding on the expression levels of NF-jB and phospo-p38 proteins in tumor tissues. Immunohistochemical and immunoblot analysis of tumor tissues showed that CP-fed mice exhibit decreased levels
of phospo-NF-jB and phospo-p38 (MAPK; Fig. 4g). CP-feeding was observed to significantly decrease activation of PI3K/
Akt signaling in tumors (Fig. 4h). These data suggest that
CP-feeding by inhibiting the Kras/Akt/NF-jB signaling network induces apoptosis and reduces the tumorigenicity of
Kras-activated PDE cells under in vivo conditions.
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Epicatechin is bioavailable to animals after CP-feeding
LC–MS/MS analysis of CP established epicatechin as the
major polyphenol (Fig. 1a). We asked if epicatechin is physiologically available to mice after CP consumption. We measured serum-epicatechin levels at time points 0.0, 0.5, 15.0,
30.0 and 180.0 min in mice receiving one-time oral feeding
of CP (100 mg/kg). Epicatechin levels were observed to be
reaching to its peak value at 15 min after CP-feeding
(Figs. 5a and 5b). Epicatechin levels were found to be detectable upto 24-hr post-CP consumption (data not shown).
Discussion
Ras mutation leads to persistent effecter pathways activation
and plays a critical role in growth and proliferation of neoplastic cells, thus has emerged as an important target for
novel therapies. There are not many well-defined genetic
human cell culture models for the study of Kras-driven
PDAC development.18,33–37 Recently, Campbell et al. developed a novel cell-based in vitro model of Kras-driven PDAC
development.9 The advantage of this cell system is that the
immortalized normal PDE cells (E6E7-st) do not exhibit
tumorigenic properties. However, their derivatives that harbor Kras mutation (E6E7-Kras-st) showed robust tumorigenic
activities.9 Moreover, these cells are recently established and
are likely to have much fewer mutations compared with
other cell lineage established by others.33–37 In this study, we
have employed this cell-based model to test our hypothesis.
This would be the first study to establish the utility of this
model for chemotherapeutic and chemoprevention studies.
Although pharmaceutical agents that target critical steps in
Kras pathway (such as farnesyl transferase inhibitors) have
been developed and tested in clinical settings, these have
Cancer Therapy
(Fig. 4b). Tumors from three animals from control and treated
group were excised at the 42nd day postfeeding when 100%
control (water-fed) animals reached the tumor volume of
1,000 mm3. Rest of the animals in treated group remained
on the protocol, until they cross the preset end point, that is,
tumor volume of 1,000 mm3.
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1730
proven not to be sufficient in preventing and treating PDAC.8
This is due to the activation of other signaling pathways,
which co-operate with Kras to enhance the growth of PDE tumor cells.38–40 Therefore, there is an unmet need to adopt
strategies that target multiple pathways. We believe that CP
possesses the property to target multiple signaling pathways.13–
17
Our argument about the potential of CP is strengthened by
the fact that CP is (i) under investigation in 30 human intervention studies, (ii) nontoxic, (iii) acceptable to human, (iv)
rich in epicatechin content and (v) economically feasible.13–
17,22,23
In this study, CP and epicatechin were observed to
reduce the activated levels of Kras (GTP-bound Ras) in E6E7Ras-st cells. A stronger effect of CP than epicatechin alone was
observed, which could be due to the presence of other polyphenols (though in minimal amount) that might have caused
an additive effect. It is noteworthy that CP preferentially inhibited growth of Kras-activated PDE cells while spared normal
PDE cells. Our data are significant, because in recent years,
emphasis is on agents capable of selective/preferential elimination of cancer cells while sparing normal cells.
The inactivation/deregulation of apoptosis is central to the
induction of neoplasm development from normal cells.37 Our
data show that CP treatment causes a cell-cycle arrest of
Kras-activated PDE cells followed by apoptosis. The antiapoptotic proteins have been reported to be elevated in PDAC
patients.39 Activation of Bcl-2 and Bcl-xL is reported to rescue the premalignant and malignant cells from the apoptosis.40,41 Report shows that an imbalance between antiapoptotic proteins and proapoptotic proteins is involved in the
distinctive biologic features of adenocarcinomas of the pancreas.42 Recent clinical studies have shown the promise of
therapies directed against Bcl-2 and Bcl-xL for human pancreatic carcinoma.43,44 Our data in this context are significant, because we observed that epicatechin-rich CP induces
apoptosis and modulates Bcl-2, Bcl-xL and Bax levels in
Kras-activated cells. We suggest that CP, by causing a shift in
the Bax/Bcl-2 ratio toward apoptosis, results in the inhibition
of growth and tumorigenicity of Kras-activated PDE cells.
Ras protein is reported to regulate PI3K/Akt and MAPK
pathways.29,33–37 Report shows that 20–70% of patients with
PDAC exhibit activation of Akt.5,6,30,45 The growth-promoting potential of the Akt and MAPK pathways and its antiapoptotic properties are closely linked to the resistance of
PDAC cells to a broad spectrum of apoptotic stimuli.30,37,45
Our data are significant, because we show that CP has the
potential to inhibit PI3K/Akt and MAPK signaling in Krasactivated PDE cells both in vitro and in vivo. We argue that
CP by inhibiting Kras activity results in the inhibition of
PI3K and MAPK signaling in Kras-activated PDE cells.
Kras oncoprotein contributes to constitutive activation of
NF-jB signaling in PDAC through the activation of PI3K/
Akt and MAPK pathways.31 p65 subunit of NF-jB is
reported to be constitutively activated in 67% of PDAC
patients.31,32 A positive correlation has been shown to exist
between the activation of NF-jB and Ras-oncoprotein during
Epicatechin-rich cocoa polyphenol inhibition
the proliferation of PDAC cells.42 Studies have shown that
PDAC cells resistant to apoptosis exhibit a high-basal NF-jB
activity.7,31 It is noteworthy that treatment of CP inhibited
phosphorylation of IjBa and NF-jB protein in Kras-activated PDE cells. Because CP inhibits IjBa phosphorylation,
we speculate that the effect of CP on NF-jB/p65 is through
inhibition of phosphorylation of IjBa, which in turn could
be regulated Ras and PI3K/Akt pathways. As Kras-driven
PI3K and NF-jB play a role in early development of PDAC,
CP intervention could be a potential strategy to prevent the
progression of this disease.
The content of epicatechin in a broad range of dark chocolates varies from 0.071 to 1.942 mg/g.38 On the basis of our
data and previously published reports, we hypothesize that
the beneficial effect of CP could be related to total epicatechin content present in it and the bioavailability of epicatechin. Epicatechin-plasma concentrations observed in this
study are in agreement with the previously published studies
conducted in animals and human subjects (Ref. 24 and references therein). These studies suggest that 30–50% of orally
administered epicatechin is absorbed from the digestive tract
and distributed in blood as conjugated forms.24 It is noteworthy that the antiproliferative and antitumoral properties of
polyphenols present in CP could also be related to the degree
of polymerization of conjugated forms in the plasma.24
In this study, infusion of CP three times in a week
resulted in a significant increase in tumor-free survival in
mice. Keeping in view this study and the findings of others,
we suggest that the daily consumption of small amounts of
polyphenols from cocoa or cocoa-based functional foods in
conjunction with usual dietary intake of flavonoids from
mixed food sources can result in an increase of plasma flavanol concentrations, which collectively may (i) contribute
to the protection against pathologies including PDAC development, (ii) prolong the survival and (iii) improve the quality of life of PDAC patients that could have immediate clinical importance.
To summarize, our findings established the efficacy of CP
against Kras-activated PDAC cells growth and identified the
underlying mechanisms. These observations warrant further
in vivo efficacy studies in models that mimic progressive
forms of human PDAC. Further in depth, in vivo studies are
warranted to verify this suggestion and are currently under
investigation in our laboratory. Our study has far reaching
health relevance as cocoa polyphenol-based foods could be
projected as functional foods that in addition to providing
nutrition would provide preventive therapeutic value against
the development of cancer.
Acknowledgements
This study was supported by departmental funds from The Hormel Institute
to the corresponding author. The authors highly acknowledge the technical
assistance of Neelofar Jan and Mohammad Naime. We are thankful to the
research division of Barry Callebaut Innovations (Lebbeke, Belgium) for
providing ACTICOA-grade cocoa polyphenol powder.
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1731
Siddique et al.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Siegel R, Ward E, Brawley O, Jemal A. Cancer
statistics. CA Cancer J Clin 2011;61:212–36.
Hruban RH, Maitra A, Kern SE, Goggins M.
Precursors to pancreatic cancer. Gastroenterol
Clin North Am 2007;36:831–49.
Welsch T, Kleeff J, Friess H. Molecular
pathogenesis of pancreatic cancer: advances and
challenges. Curr Mol Med 2007;7:504–21.
Takaori K. Current understanding of precursors
to pancreatic cancer. J Hepatobiliary Pancreat
Surg 2007;14:217–23.
Deramaudt T, Rustgi AK. Mutant KRAS in the
initiation of pancreatic cancer. Biochim Biophys
Acta 2005;1756:97–101.
Cicenas J. The potential role of Akt
phosphorylation in human cancers. Int J Biol
Markers 2008;23:1–9.
Holcomb B, Yip-Schneider M, Schmidt CM. The
role of nuclear factor kappaB in pancreatic cancer
and the clinical applications of targeted therapy.
Pancreas 2008;36:225–35.
Furukawa T. Molecular targeting therapy for
pancreatic cancer: current knowledge and
perspectives from bench to bedside.
J Gastroenterol 2008;43:905–11
Campbell PM, Groehler AL, Lee KM, Ouellette
MM, Khazak V, Der CJ. K-Ras promotes growth
transformation and invasion of immortalized
human pancreatic cells by Raf and
phosphatidylinositol 3-kinase signaling. Cancer
Res 2007;67:2098–106.
Stan SD, Singh SV, Brand RE. Chemoprevention
strategies for pancreatic cancer. Nat Rev
Gastroenterol Hepatol 2010;7:347–56.
Doucas H, Garcea G, Neal CP, Manson MM,
Berry DP. Chemoprevention of pancreatic cancer:
a review of the molecular pathways involved, and
evidence for the potential for chemoprevention.
Pancreatology 2006;6:429–39.
Jew S, AbuMweis SS, Jones PJ. Evolution of the
human diet: linking our ancestral diet to modern
functional foods as a means of chronic disease
prevention. J Med Food 2009;12:925–34.
Rimbach G, Melchin M, Moehring J, Wagner AE.
Polyphenols from cocoa and vascular health: a
critical review. Int J Mol Sci 2009;10:4290–309.
Cienfuegos-Jovellanos E, Qui~
nones Mdel M,
Muguerza B, Moulay L, Miguel M, Aleixandre A.
Antihypertensive effect of a polyphenol-rich
cocoa powder industrially processed to preserve
the original flavonoids of the cocoa beans. J Agric
Food Chem 2009;57:6156–62.
Taubert D, Berkels R, Roesen R, Klaus W.
Chocolate and blood pressure in elderly
individuals with isolated systolic hypertension.
JAMA 2003;290:1029–30.
Grassi D, Necozione S, Lippi C, Croce G, Valeri
L, Pasqualetti P, Desideri G, Blumberg JB, Ferri
C. Cocoa reduces blood pressure and insulin
resistance and improves endothelium-dependent
vasodilation in hypertensives. Hypertension 2005;
46:398–405.
Buijsse B, Feskens EJ, Kok FJ, Kromhout D.
Cocoa intake, blood pressure, and cardiovascular
mortality: the Zutphen Elderly Study. Arch Intern
Med 2006;166:411–7.
Lee KM, Nguyen C, Ulrich AB, Pour PM,
Ouellette MM. Immortalization with telomerase
of the Nestin-positive cells of the human
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
pancreas. Biochem Biophys Res Commun 2003;
301:1038–44.
Murtaza I, Saleem M, Adhami VM, Hafeez BB,
Mukhtar H. Suppression of cFLIP by lupeol, a
dietary triterpene, is sufficient to overcome
resistance to TRAIL-mediated apoptosis in
chemoresistant human pancreatic cancer cells.
Cancer Res 2009;69:1156–65.
Saleem M, Kaur S, Kweon MH, Adhami VM,
Afaq F, Mukhtar H. Lupeol, a fruit and vegetable
based triterpene, induces apoptotic death of
human pancreatic adenocarcinoma cells via
inhibition of Ras signaling pathway.
Carcinogenesis 2005;26:1956–64.
Siddique HR, Mishra SK, Karnes RJ, Saleem M.
Lupeol, a novel androgen receptor inhibitor:
implications in prostate cancer therapy. Clin
Cancer Res 2011;17:5379–91.
Saleem M, Murtaza I, Tarapore RS, Suh Y,
Adhami VM, Johnson JJ, Siddiqui IA, Khan N,
Asim M, Hafeez BB, Shekhani MT, Li B, et al.
Lupeol inhibits proliferation of human prostate
cancer cells by targeting beta-catenin signaling.
Carcinogenesis 2009;30:808–17.
Saleem M, Murtaza I, Witkowsky O, Kohl AM,
Maddodi N. Lupeol triterpene, a novel diet-based
microtubule targeting agent: disrupts survivin/
cFLIP activation in prostate cancer cells. Biochem
Biophys Res Commun 2009;388:576–82.
Bisson JF, Hidalgo S, Rozan P, Messaoudi M.
Preventive effects of ACTICOA powder, a cocoa
polyphenolic extract, on experimentally induced
prostate hyperplasia in Wistar-Unilever rats. J
Med Food 2007;10:622–7.
Bisson JF, Hidalgo S, Rozan P, Messaoudi M.
Therapeutic effect of ACTICOA powder, a cocoa
polyphenolic extract, on experimentally induced
prostate hyperplasia in Wistar-Unilever rats. J
Med Food 2007;10:628–35.
Saleem M, Adhami VM, Zhong W, Longley BJ,
Lin CY, Dickson RB, Reagan-Shaw S, Jarrard DF,
Mukhtar H. A novel biomarker for staging
human prostate adenocarcinoma: overexpression
of matriptase with concomitant loss of its
inhibitor, hepatocyte growth factor activator
inhibitor-1. Cancer Epidemiol Biomarkers Prev
2006;15:217–27.
Elmore S. Apoptosis: a review of programmed
cell death. Toxicol Pathol 2007;35:495–516.
Wolff RA. Chemoprevention for pancreatic cancer.
Int J Gastroint Cancer 2003;33:27–41.
Mohammed A, Janakiram NB, Li Q, Madka V,
Ely M, Lightfoot S, Crawford H, Steele VE, Rao
CV. The epidermal growth factor receptor
inhibitor gefitinib prevents the progression of
pancreatic lesions to carcinoma in a conditional
LSL-KrasG12D/þ transgenic mouse model.
Cancer Prev Res (Phila) 2010;3:1417–26.
Nair PN, De Armond DT, Adamo ML, Strodel
WE, Freeman JW. Aberrant expression and
activation of insulin-like growth factor-1 receptor
(IGF-1R) are mediated by an induction of IGF1R promoter activity and stabilization of IGF-1R
mRNA and contributes to growth factor
independence and increased survival of the
pancreatic cancer cell line MIA-PaCa-2. Oncogene
2001;20:8203–14.
Murtaza I, Adhami VM, Hafeez BB, Saleem M,
Mukhtar H. Fisetin, a natural flavonoid,
C 2011 UICC
Int. J. Cancer: 131, 1720–1731 (2012) V
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
targets chemoresistant human pancreatic
cancer AsPC-1 cells through DR3-mediated
inhibition of NF-kappaB. Int J Cancer 2009;
125:2465–73.
Lampe E, Heinrich M, Walczak H, Kalthoff H.
CD95 and TRAIL receptor-mediated activation of
protein kinase C and NF-kappaB contributes to
apoptosis resistance in ductal pancreatic
adenocarcinoma cells. Oncogene 2001;20:
4258–426
Furukawa T, Duguid WP, Rosenberg L, Viallet J,
Galloway DA, Tsao MS. Long-term culture and
immortalization of epithelial cells from normal
adult human pancreatic ducts transfected by the
E6E7 gene of human papilloma virus 16. Am J
Pathol 1996;148:1763–70.
Jesnowski R, Muller P, Schareck W, Liebe S, Lohr
M. Immortalized pancreatic duct cells in vitro
and in vivo. Ann N Y Acad Sci 1999;880:50–65.
Liu N, Furukawa T, Kobari M, Tsao MS.
Comparative phenotypic studies of duct epithelial
cell lines derived from normal human pancreas
and pancreatic carcinoma. Am J Pathol 1998;153:
263–9.
Ouyang H, Mou Lj, Luk C, Liu N, Karaskova J,
Squire J, Tsao MS. Immortal human pancreatic
duct epithelial cell lines with near normal
genotype and phenotype. Am J Pathol 2000;157:
1623–31.
Qian J, Niu J, Li M, Chiao PJ, Tsao MS. In vitro
modeling of human pancreatic duct epithelial cell
transformation defines gene expression changes
induced by K-ras oncogenic activation in
pancreatic carcinogenesis. Cancer Res 2005;65:
5045–53.
Cooper KA, Campos-Gimenez E, Jimenez Alvarez
D, Nagy K, Donovan JL, et al. Rapid reversed
phase ultra-performance liquid chromatography
analysis of the major cocoa polyphenols and interrelationships of their concentrations in chocolate. J
Agric Food Chem 2007;55:2841–7.
Brown J, Attardi L. The role of apoptosis in
cancer development and treatment response.
Nature Rev 2005;5:231–7.
Miyamoto Y, Hosotani R, Wada M, Lee JU,
Koshiba T, Fujimoto K, Tsuji S, Nakajima S, Doi
R, Kato M, Shimada Y, Imamura M.
Immunohistochemical analysis of Bcl-2, Bax, BclX, and Mcl-1 expression in pancreatic cancers.
Oncology 1999;56:73–82.
Xu Z, Friess H, Solioz M, Aebi S, Korc M, Kleeff
J, Büchler MW. Bcl-xL antisense oligonucleotides
induce apoptosis and increase sensitivity of
pancreatic cancer cells to gemcitabine. Int J
Cancer 2001;94:268–74.
Rajalingam K, Schreck R, Rapp UR, Albert S. Ras
oncogenes and their downstream targets. Biochim
Biophys Acta 2007;1773:1177–95.
Pino SM, Xiong HQ, McConkey D, Abbruzzese
JL. Novel therapies for pancreatic
adenocarcinoma. Curr Oncol Rep 2004;6:199–206.
Okamoto K, Murawaki Y. The therapeutic
potential of RNA interference: novel approaches
for cancer treatment. Curr Pharm Biotechnol, in
press; PMID: 21605072.
Ottenhof NA, de Wilde RF, Maitra A, Hruban
RH, Offerhaus GJ. Molecular characteristics of
pancreatic ductal adenocarcinoma. Pathol Res Int
2011;2011:620601.
Cancer Therapy
References