ve
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eDepartment of Internal Medicine VA Nebraska-Western Iowa Health Care System and University of Nebraska Medical Center, Omaha, NE 68198-5870, USA
diagnosis, PC is already in advanced stages (i.e. III and IV) and is
resistant to conventional chemotherapy and radiotherapy [3].
Interestingly, even patients diagnosed with stage I PC that have
the option to undergo surgery have a 5-year overall survival of
approximately 20%, a clear indication of the general failure of cur-
phase III clinical trial reported an improved overall survival of
PC patients treated with a four-drug chemotherapy regimen com-
prising fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLF-
IRINOX). Nevertheless, a major disadvantage of this novel
treatment was its related toxicity, which was noticeably high
when compared to PC patients treated with gemcitabine alone.
Therefore, novel, alternative PC therapeutics must not only im-
prove the prognosis of PC patients but also minimize any possible
toxicity-related side effects that will interfere with the quality of
life of PC patients.
⇑ Corresponding author at: Department of Internal Medicine VA Nebraska-
Western Iowa Health Care System and University of Nebraska Medical Center,
Omaha, NE 68198-5870, USA. Tel.: +1 402 559 5455; fax: +1 402 559 6650.
Cancer Letters 323 (2012) 29–40
Contents lists available at
Cancer L
.e l
E-mail address: sbatra@unmc.edu (S.K. Batra).
these parameters led to a decrease in tumorigenicity and metastasis of orthotopically implanted pancre-
atic tumors, indicating promising characteristics of the natural product against this lethal disease.
� 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
The overall 5-year survival rate for pancreatic cancer (PC) pa-
tients was 5.5% for the period of 2001–2007, according to the Na-
tional Cancer Institute (NCI), a statistic that has not varied
significantly for over the last four decades [1]. In 2012, it is esti-
mated that 43,920 new PC cases will be diagnosed and approxi-
mately 85% of these (i.e. 37,390) will succumb to the disease [2].
The main reason behind the poor prognosis of PC patients is the
insidious and sporadic nature of the disease, which is often pre-
sented with no specific early clinical symptoms. By the time of
rent standard treatments for each stage of PC [4,5]. What is even
more alarming, are the statistics that predict a 55% increase in
the expected number of new PC cases by 2030 [6]. Thus, immediate
progress must be made in the prevention, early diagnosis, and sys-
temic treatments against this lethal disease.
Gemcitabine has been the standard line of treatment for PC
patients for over a decade and is associated with a median patient
survival of 5.4 months [7]. Over all these years, numerous clinical
efforts have been devoted to improve PC chemotherapy outcomes,
but unfortunately no significant improvements have been re-
ported apart from a clinical trial reported in May of 2011 [8]. This
a r t i c l e i n f o
Article history:
Received 19 January 2012
Received in revised form 25 February 2012
Accepted 26 March 2012
Keywords:
Pancreatic cancer
Therapy
Cancer metabolism
Natural product
0304-3835/$ - see front matter � 2012 Elsevier Irelan
http://dx.doi.org/10.1016/j.canlet.2012.03.031
a b s t r a c t
Pancreatic tumors are resistant to conventional chemotherapies. The present study was aimed at evalu-
ating the potential of a novel plant-derived product as a therapeutic agent for pancreatic cancer (PC). The
effects of an extract from the tropical tree Annona Muricata, commonly known as Graviola, was evaluated
for cytotoxicity, cell metabolism, cancer-associated protein/gene expression, tumorigenicity, and meta-
static properties of PC cells. Our experiments revealed that Graviola induced necrosis of PC cells by inhib-
iting cellular metabolism. The expression of molecules related to hypoxia and glycolysis in PC cells (i.e.
HIF-1a, NF-jB, GLUT1, GLUT4, HKII, and LDHA) were downregulated in the presence of the extract. In
vitro functional assays further confirmed the inhibition of tumorigenic properties of PC cells. Overall,
the compounds that are naturally present in a Graviola extract inhibited multiple signaling pathways that
regulate metabolism, cell cycle, survival, and metastatic properties in PC cells. Collectively, alterations in
cDepartment of Environmental, Agricultural & Occupational Health, Omaha, NE 68198-5870, USA
dDepartment of Pathology and Microbiology, Omaha, NE 68198-5870, USA
Graviola: A novel promising natural-deri
and metastasis of pancreatic cancer cells
cell metabolism
María P. Torres a,b, Satyanarayana Rachagani a, Vinee
Erik D. Moore a, Sonny L. Johansson b,d, Pankaj K. Sin
aDepartment of Biochemistry and Molecular Biology, Omaha, NE 68198-5870, USA
b Eppley Institute for Research in Cancer and Allied Diseases, Omaha, NE 68198-5870, U
journal homepage: www
d Ltd. All rights reserved.
d drug that inhibits tumorigenicity
vitro and in vivo through altering
rohit b, Poomy Pandey c, Suhasini Joshi a,
a,b, Apar K. Ganti e, Surinder K. Batra a,b,d,⇑
SciVerse ScienceDirect
etters
sevier .com/locate /canlet
r Le
It is well known that an increased consumption of fruits and
vegetables is associated with a reduced risk of most cancers,
including PC [9]. For this reason, the potential of natural products
in PC therapies has been widely investigated [10]. While some of
these compounds have undergone clinical testing (i.e. curcumin,
genistein) and have demonstrated some activity against PC, the
poor bioavailability in patients minimizes their therapeutic effi-
cacy. However, as compared with conventional chemotherapeutic
drugs, the major benefit of these therapies is the apparent lack of
toxicities to healthy tissues. These facts attracted our attention to
find alternative, natural-derived chemotherapeutic drugs in order
to improve the prognosis of PC patients.
Traditionally, the leaves from the tropical tree Annona Muricata,
also known as Graviola or Soursop, have been used for a wide
range of human diseases including inflammatory conditions, rheu-
matism, neuralgia, diabetes, hypertension, insomnia, cystitis, para-
sitic infections, and cancer [11]. The major bioactive components
that have been extracted from different parts of the plant are
known as Annonaceous acetogenins. These are derivatives of long
chain (C35 or C37) fatty acids derived from the polyketide pathway
[12] that are selectively toxic to cancer cells, including multidrug-
resistant cancer cell lines [13–17]. Annonaceous acetogenins in-
duce cytotoxicity by inhibiting the mitochondrial complex I, which
is involved in ATP synthesis [14]. As cancer cells have a higher de-
mand for ATP than the normal cells, mitochondrial complex I
inhibitors have potential in cancer therapeutics.
A few in vivo studies involving A. Muricata have been reported.
Among these, two reports have shown the ability of the leaf extract
to regenerate pancreatic islet b cells in diabetic rats [18,19]. These
studies suggest an additional benefit of the natural product against
PC given that diabetes has been classified as a risk factor of the
malignant disease [20]. More recently, one study analyzing the
anti-tumor efficacy of A. Muricata was published [21]. The extract
had a direct anti-tumorigenic effect on breast cancer cells by
downregulating the expression of the epidermal growth factor
receptor (EGFR). Although this study demonstrates the potential
anti-tumorigenic properties of Graviola, the doses used in the
experimental design were not properly controlled. The mice were
fed with the extract mixed in the diet and the exact amount in-
gested by each animal could not be estimated accurately.
Although a few in vitro reports have shown the cytotoxic charac-
teristics of Graviola against various cancer cell lines, including PC
cells [12], the comprehensive in vivo effects and mechanistic scien-
tific studies are still lacking. To our knowledge, the studies reported
herein are the first to indicate that Graviola extract has promising
characteristics for PC therapeutics. Comprehensive in vitro and
in vivo studies in various PC cell lines revealed that this natural prod-
uct has inhibited multiple signaling pathways that regulate metab-
olism, cell cycle, survival, and metastatic properties of PC cells.
2. Materials and methods
2.1. Graviola extract
Graviola supplement capsules were purchased from Raintree (Carson City, NV).
The capsules consisted of 100% pure, finely milled Graviola leaf/stem powder with
no binders or fillers. The capsule contents were suspended in DMSO (100 mg/mL).
After incubating for 5 min, the suspension was centrifuged and the supernatant (i.e.
extract) was filtered to remove any remaining particles. Subsequent dilutions were
prepared in Dulbecco’s modification of Eagle’s medium (DMEM) supplemented
with 10% of fetal bovine serum (FBS). Stock solutions and respective dilutions were
freshly prepared prior to treatment.
2.2. Cell culture
The metastatic PC cell lines FG/COLO357 and CD18/HPAF were purchased from
30 M.P. Torres et al. / Cance
the American Type Culture Collection (ATCC). Before performing experiments, the
PC cell lines were authenticated by short tandem repeat analysis. It was ensured
that PC cells were used at fewer than 20 passages after purchase from ATCC. Cells
were cultured in DMEM medium supplemented with 10% FBS and antibiotics
(100 lg/mL penicillin and 100 lg/mL streptomycin). The cells were maintained at
37 �C and 5% CO2 in a humidified atmosphere.
2.3. Antibodies
The antibodies for phospho-ERK1/2, total ERK, phospho-Akt (Ser 473), total Akt,
NF-jB, and Caspase-3 were purchased from Cell Signaling Technology (Danvers,
MA). The antibodies for Cyclin-D1, phospho-FAK (Tyr 925), and total FAK were ob-
tained from Santa Cruz Biotechnology (Santa Cruz, CA). The b-actin and b-Tubulin
antibodies were obtained from Sigma Aldrich (St. Louis, MO), whereas the HIF-1a
antibody was purchased from BD Biosciences (San Jose, CA). The MUC4 monoclonal
antibody (8G7) used in these studies was developed by our group [22]. MMP9 anti-
body was obtained from a hybridoma cell supernatant kindly provided by
Dr. Rakesh Singh at UNMC. The secondary antibodies used for western blot analyses
were the ECL™ anti-mouse and anti-rabbit IgG conjugated to horseradish peroxi-
dase (GE healthcare, UK). Fluorescein isothiocyanate (FITC) conjugated-anti-mouse
and Alexa Fluor conjugated anti-mouse antibodies were obtained from Invitrogen
(Carlsbad, CA).
2.4. Cytotoxicity assay
To determine the cytotoxicity of Graviola extract on PC cells, 1 � 104 cells were
seeded per well on a 96-well plate in DMEM supplemented with 10% FBS and anti-
biotics. After overnight incubation, different concentrations (10–200 lg/mL) of the
extract were added into triplicate wells. After 48 h, the media was replaced with
fresh media containing thiazolyl blue tetrazolium bromide (MTT) reagent (Sigma
Aldrich, St. Louis, MO). After 4 h incubation at 37 �C in 5% CO2 in humidified atmo-
sphere, the media was replaced with 100 lL of DMSO and the corresponding cyto-
toxicity values were calculated (k = 540 nm). The experiment was repeated at least
three times.
2.5. Western blot analysis
For protein analysis, 0.5 � 106 of PC cells were seeded on each well of a six-well
plate in DMEM supplemented with 10% FBS and antibiotics. After overnight incuba-
tion, fresh solutions of Graviola (0–200 lg/mL) were prepared and added to the
respective wells. Cells incubated with the corresponding amount of DMSO present
in the highest concentrated solution of Graviola were used as a negative control
(0 lg/mL). After 48 h of incubation with the extract, protein lysates were isolated
and prepared for western blot analysis, as previously described [23].
2.6. Real-time PCR
The transcripts levels of the glucose transporters GLUT1 and GLUT4, the glyco-
lytic enzymes hexokinase II (HKII) and lactate dehydrogenase A (LDHA), and the
mucin glycoprotein MUC4 in PC cells were determined after treatment with Gravi-
ola extract by real-time PCR. 0.5 � 106 cells were seeded in each well of a six-well
plate in complete media. After overnight incubation, fresh solutions of Graviola
extract (50 and 100 lg/mL) were prepared and cells were incubated for 48 h. Sub-
sequently, cDNA was synthesized from purified RNA and real-time PCR was carried
out as has been described by previous studies [23]. The sequences of the gene-spe-
cific primers used were:
GLUT1: F 50-GCCATGGAGCCCAGCAGCAA-30; R 50-CGGGGACTCTCGGGGCAGAA-30
GLUT4: F 50-GCCTGTGGCCACTGCTCCTG-30; R 50-GGGGTCTCTGGGCCGGGTAG-30
HKII: F 50-GTCATCCCCTTGTGTCAGAG-30; R 50-CTTCATTAGTGTCCCCATCCTG-30
LDHA: F 50-CCAGTGTGCCTGTATGGAGTG-30; R 50-GCACTCTCAACCACCTGCT
TG-30
MUC4: F 50-GTGACCATGGAGGCCAGTG-30; R 50-TCATGCTCAGGTGTCCACAG-30
2.7. Glucose uptake
Glucose-uptake rate was assayed by utilizing [3H] 2-deoxyglucose ([3H] 2-DG).
5 � 104 PC cells were seeded per well in a 24-well plate. 12 h later, the cells were
treated with Graviola extract (10 and 50 lg/mL) for 48 h. The cells were then
starved for glucose for 2 h and incubated for 20 min with 2 lCi [3H] 2-DG. Subse-
quently, cells were lysed with 1% SDS and the lysates were counted for [3H] by uti-
lizing a scintillation counter. Cells treated with labeled and excess unlabeled 2-DG
were used as controls to set a baseline for non-specific [3H] uptake. The results were
normalized to the cell counts for treated and untreated groups. Glucose uptake was
normalized with that of the control cells (0 lg/mL) and it is presented as the mean
values ± standard error from experiments performed in triplicate.
2.8. ATP quantification
tters 323 (2012) 29–40
The CellTiter-Glo� Luminescent Cell Viability Assay (Promega, Madison, WI)
was used to measure the ATP content in the cells. Briefly, 1 � 104 PC cells were
seeded in each well of an opaque 96-well plate. Cells were seeded for both ATP
On the necropsy day, pancreatic tumors from the different treatment groups
For histopathological and IHC analyses, the tumor tissues were fixed in 10% For-
malin for 48 h. The tumors were embedded in paraffin and 5 lm sections were cut
3.1. Graviola extract induces cytotoxicity of pancreatic cancer cells
r Le
quantification and protein concentration estimation. Starting the next day, the cells
were incubated with Graviola extract-containing media for 48 h. Subsequently, the
instructions of the manufacturer for ATP quantification were followed and lumines-
cence was measured on a Synergy™Mx Luminescent Plate Reader (BioTek,
Winooski, VT). Data is presented as the mean value for samples in triplicates, nor-
malized with the protein content for each treatment, as determined by utilizing mi-
cro-BCA protein estimation kit.
2.9. Detection and quantification of apoptosis and necrosis
To quantify the number of PC cells undergoing apoptosis and necrosis after
being incubated with Graviola extract, the Annexin-V-FLUOS staining kit (Roche
Diagnostics, Indianapolis, IN) was used. PC cells were seeded and treated with Gra-
viola extract as described above. After 48 h of treatment with Graviola extract, the
instructions of the manufacturer were followed for staining cells for flow cytometry
analysis. The experiment was repeated three times.
2.10. Cell cycle analysis
PC cells were synchronized at the G1/S phase using a double thymidine block.
After seeding cells in 100 cm2 Petri dishes, thymidine (2 mM) was added for 12 h.
After washing cells with serum-free media, the cells were released from thymidine
block by culturing in fresh medium containing 24 mM 2-deoxycytidine for 9 h.
Then, cells were washed and incubated once more with thymidine (2 mM) for
14 h. Subsequently, the cells were released from the second thymidine block and
the respective treatment prepared in complete media was added for 48 h. For cell
cycle analysis, cells were trypsinized and washed with PBS after the duration of
the treatment. Cells were then fixed in 70% ethanol at 4 �C for 1 h. After washing,
cells were incubated with Telford reagent (EDTA, RNAse A, propidium iodide, Triton
X-100 in PBS) at 4 �C and analyzed by flow cytometry on the next day.
2.11. Confocal microscopy
For confocal analysis, 2 � 105 PC cells were seeded on sterilized round glass
cover slips. After overnight incubation, Graviola extract (0, 50 and 100 lg/mL)
was added to the cells, followed by a 48 h incubation. For the detection of reactive
oxygen species (ROS), Graviola extract-treated PC cells were incubated with 1 lM
20-70-Dichlorofluorescein diacetate (DCFH-DA) (Sigma Aldrich, St. Louis, MO) for
15 min. After three washes with PBS, glass cover slips were mounted on glass slides
and visualized by confocal microscopy. For b-tubulin and MUC4 confocal analysis,
details of the procedure are published elsewhere [23]. Finally, to visualize the
arrangement of actin filaments in Graviola extract-treated cells, the cells were
stained with fluorescent phallotoxins (Invitrogen, Carlsbad, CA). The instructions
of the manufacturer were followed for formaldehyde-fixed cells. Post-staining,
the glass cover slips were mounted with Vectashield medium (Vector Laboratories,
Burlingame, CA). LSM 510 microscope, a laser scanning confocal microscope (Carl
Zeiss GmbH, Thornwood, NY) was utilized to image the cells in the respective chan-
nels at a magnification of 630�.
2.12. Wound healing assay
For wound healing assays, 3 � 106 of PC cells were seeded in 60 mm petri dishes
in DMEM media supplemented with 10% FBS and antibiotics. After overnight incu-
bation, an artificial wound was induced on 100% confluent PC cell monolayers using
a sterile pipette tip. Graviola extract-containing (0, 50, 100 lg/mL) media solutions
were then added to the respective treatment plate. Images (40�) were captured
immediately after adding Graviola extract (0 h) and after 24 h of treatment, by a
light microscope. The motility of the cells across the wound was visualized in each
treatment group.
2.13. Motility assay
The effect of Graviola extract on the migration of PC cells was also analyzed by a
transwell migration assay. FG/COLO357 cells (0.5 � 106) were suspended in Gravi-
ola extract-containing (0–100 lg/mL) 1% FBS-DMEM media and seeded for 48 h in
8 lm pore size polyethylene terephthalate (PET) membranes (Becton Dickinson,
San Jose, CA). DMEM supplemented with 10% FBS was added at the bottom of each
well and after 48 h of incubation, the cells that migrated to the bottom of the PET
membrane were stained with Diff-Quick cell staining kit (Dade Behring Inc., New-
ark, DE). The number of migrated cells was quantified by performing cell counts of
10 random fields at 100� magnification. The results are presented as the average
number of cells in one field.
2.14. In vivo tumorigenicity studies
The effect of Graviola extract on pancreatic tumor growth was evaluated on
M.P. Torres et al. / Cance
orthotopic tumor xenografts. 6–8 week old female athymic immunodeficient mice
were purchased from the Animal Production Area of the NCI/Frederick Cancer Re-
The PC cells FG/COLO357 and CD18/HPAF were incubated for
48 h with different concentrations of Graviola extract. The results
from the MTT cytotoxicity assay indicated a progressive decrease
in cell viability with the successive increase in the concentrations
of the extract (Fig. 1A). After 48 h of treatment, the resulting
IC50 of Graviola extract on FG/COLO357 and CD18/HPAF cells
was 200 and 73 lg/mL, respectively and the results indicated that
CD18/HPAF cell line is more sensitive to the Graviola extract than
the FG/COLO357 cell line (Fig. 1B).
It is well known that the activation of the extracellular signal-
regulated kinase (ERK) and the phosphatidylinositol 30kinase
(PI3K/Akt) pathways play a crucial role in the proliferation and sur-
vival of PC [26] and inhibition of these pathways leads to the inhi-
bition of pancreatic tumor growth [27,28]. The present study
reveal