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Selective Growth Inhibition of Human Breast Cancer Cells by Graviola Fruit Extract In Vitro and In Vivo Involving Downregulation of EGFR Expression

Taylor & Francis
Nutrition and Cancer
Authors:
  • Takeda pharmaceuticals International Co.
  • Virginia Tech

Abstract and Figures

The epidermal growth factor receptor (EGFR) is an oncogene frequently overexpressed in breast cancer (BC), and its overexpression has been associated with poor prognosis and drug resistance. EGFR is therefore a rational target for BC therapy development. This study demonstrated that a graviola fruit extract (GFE) significantly downregulated EGFR gene expression and inhibited the growth of BC cells and xenografts. GFE selectively inhibited the growth of EGFR-overexpressing human BC (MDA-MB-468) cells (IC(50) = 4.8 μg/ml) but had no effect on nontumorigenic human breast epithelial cells (MCF-10A). GFE significantly downregulated EGFR mRNA expression, arrested cell cycle in the G0/G1 phase, and induced apoptosis in MDA-MB-468 cells. In the mouse xenograft model, a 5-wk dietary treatment of GFE (200 mg/kg diet) significantly reduced the protein expression of EGFR, p-EGFR, and p-ERK in MDA-MB-468 tumors by 56%, 54%, and 32.5%, respectively. Overall, dietary GFE inhibited tumor growth, as measured by wet weight, by 32% (P < 0.01). These data showed that dietary GFE induced significant growth inhibition of MDA-MB-468 cells in vitro and in vivo through a mechanism involving the EGFR/ERK signaling pathway, suggesting that GFE may have a protective effect for women against EGFR-overexpressing BC.
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Selective Growth Inhibition of Human Breast Cancer
Cells by Graviola Fruit Extract In Vitro and In Vivo
Involving Downregulation of EGFR Expression
Yumin Dai a , Shelly Hogan b , Eva M. Schmelz c , Young H. Ju c , Corene Canning d & Kequan
Zhou d
a Department of Food Science and Technology, Virginia Tech, Blacksburg, Virginia, USA
b Montana State University, Bozeman, Montana, USA
c Department of Human Nutrition, Foods and Exercise, Virginia Tech, Blacksburg, Virginia,
USA
d Department of Nutrition and Food Science, Wayne State University, Detroit, Michigan, USA
Available online: 22 Jun 2011
To cite this article: Yumin Dai, Shelly Hogan, Eva M. Schmelz, Young H. Ju, Corene Canning & Kequan Zhou (2011): Selective
Growth Inhibition of Human Breast Cancer Cells by Graviola Fruit Extract In Vitro and In Vivo Involving Downregulation of
EGFR Expression, Nutrition and Cancer, 63:5, 795-801
To link to this article: http://dx.doi.org/10.1080/01635581.2011.563027
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Nutrition and Cancer, 63(5), 795–801
Copyright C
2011, Taylor & Francis Group, LLC
ISSN: 0163-5581 print / 1532-7914 online
DOI: 10.1080/01635581.2011.563027
Selective Growth Inhibition of Human Breast Cancer Cells
by Graviola Fruit Extract In Vitro and In Vivo Involving
Downregulation of EGFR Expression
Yumin Dai
Department of Food Science and Technology, Virginia Tech, Blacksburg, Virginia, USA
Shelly Hogan
Montana State University, Bozeman, Montana, USA
Eva M. Schmelz and Young H. Ju
Department of Human Nutrition, Foods and Exercise, Virginia Tech, Blacksburg, Virginia, USA
Corene Canning and Kequan Zhou
Department of Nutrition and Food Science, Wayne State University, Detroit, Michigan, USA
The epidermal growth factor receptor (EGFR) is an oncogene
frequently overexpressed in breast cancer (BC), and its overexpres-
sion has been associated with poor prognosis and drug resistance.
EGFR is therefore a rational target for BC therapy development.
This study demonstrated that a graviola fruit extract (GFE) sig-
nificantly downregulated EGFR gene expression and inhibited the
growth of BC cells and xenografts. GFE selectively inhibited the
growth of EGFR-overexpressing human BC (MDA-MB-468) cells
(IC50 =4.8 µg/ml) but had no effect on nontumorigenic human
breast epithelial cells (MCF-10A). GFE significantly downregu-
lated EGFR mRNA expression, arrested cell cycle in the G0/G1
phase, and induced apoptosis in MDA-MB-468 cells. In the mouse
xenograft model, a 5-wk dietary treatment of GFE (200 mg/kg diet)
significantly reduced the protein expression of EGFR, p-EGFR,
and p-ERK in MDA-MB-468 tumors by 56%, 54%, and 32.5%,
respectively. Overall, dietary GFE inhibited tumor growth, as mea-
sured by wet weight, by 32% (P<0.01). These data showed that
dietary GFE induced significant growth inhibition of MDA-MB-
468 cells in vitro and in vivo through a mechanism involving the
EGFR/ERK signaling pathway,suggesting that GFE may have a
protective effect for women against EGFR-overexpressing BC.
INTRODUCTION
Breast cancer (BC) remains one of the most common female
cancers (1). In 2010, there were an estimated 207,090 new cases
Submitted 28 May 2010; accepted in final form 3 February 2011.
Address correspondence Kequan Zhou, Department of Nutrition
and Food Science, Wayne State University, Detroit, MI 48202. Phone:
1 3135773444. Fax: 1 3135778616. E-mail: kzhou@wayne.edu
and 39,840 deaths from BC in the United States (2). Considering
the heterogeneity of BC and the limitations of current therapies
due to severe side effects and drug resistance, there is an urgent
need to explore alternative strategies to prevent and treat BC.
Development of novel mechanism-based nutritional agents that
could selectively target BC may offer an intriguing strategy
for controlling the disease. Accumulating evidence suggests a
strong effect of the diet or its components on BC development
and progression, either through effects on hormonal status or
via direct anti-tumor-promoting or anticarcinogenic effects (3).
Epidemiologic studies have linked greater fruit and vegetable
intake with lower risk of cancer (4). This beneficial effect is
due in part to the fact that fruits and vegetables contain fiber,
antioxidants, and other potentially antineoplastic compounds.
Specific food bioactive components, notably sulfur-containing
glucosinolates and green tea polyphenols, are associated with
reduced risk of BC (5,6).
Graviola (Annonaceous muricata L.) is an Amazon fruit tree
that grows in the tropics of North and South America and is also
known as soursop and guanabana. Leaves and stems of graviola
have been traditionally used as an herbal preparation for a vari-
ety of purported health-promoting effects, including supporting
healthy cell growth and immune function (7). Graviola fruits
have been widely consumed by indigenous people in fresh or
processed forms for centuries. However, research on this fruit is
extremely limited despite its regular consumption, and to date,
there is no published study investigating the effect of graviola
fruit on cancers.
The EGFR has been identified as a promising target for BC
therapies. Binding of a ligand such as EGF or transforming
795
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796 Y. DA I E T A L .
growth factor α(TGFα) results in a signaling cascade that pro-
duces diverse effects, including cell migration, maturation, dif-
ferentiation, metastasis, angiogenesis, and inhibition of apopto-
sis (9). High expression of EGFR is commonly considered the
main mechanism by which EGFR signaling is increased in can-
cer cells (10). EGFR or its family members are highly expressed
in a variety of human tumors, including BC (11). Its overexpres-
sion correlates inversely with estrogen receptor (ER) status in
patients with BC (12) and is associated with poor prognosis
and resistance to chemotherapy, hormone therapy, and radiation
(13). Molecular inhibition of EGFR signaling by anti-EGFR
monoclonal antibody or specific small molecule inhibitors has
shown antitumor effects in clinical trials (14). This study deter-
mined the in vitro and in vivo effect of GFE treatment on the
growth of MDA-MB-468 cells through a mechanism involving
downregulation of EFGR expression and downstream effectors
of the EGFR signaling pathway. MDA-MB-468 cells contain
an amplified EGFR gene (8) and therefore serve as an excellent
model in which to study anti-EGFR treatment.
MATERIALS AND METHODS
Materials
Methyl thiazolyl tetrazolium bromide (MTT), propidium
iodide (PI), and Triton X-100, 4,6-diamidino-2-phenylidple
(DAPI) were purchased from Sigma Chemical Company (St.
Louis, MO). An annexin V-FITC cell apoptosis kit was obtained
from Zymed Laboratories, Inc. (San Francisco, CA). Reagents
for RNA extraction and purification were purchased from Qia-
gen (Valencia, CA). Reagents for cDNA synthesis and quantita-
tive reverse transcriptase-polymerase chain reaction (qRT-PCR)
were purchased from Bio-Rad (Hercules, CA). Primers were
purchased from Integrated DNA Technologies, Inc. (Coralville,
IA). Animal diet was purchased from Dyets (Bethlehem, PA).
Graviola Fruit Extraction and Purification
The dried graviola fruit powder was obtained from Earth-
fruits (South Jordan, UT). GFE was prepared with 50% aque-
ous acetone extraction, which was subsequently filtered and
lyophilized. Approximately 100–120 mg GFE was obtained
from 10 g dried graviola fruit powder.
Cell Lines and Cell Culture
Human BC MDA-MB-468, MDA-MB-231, and MCF-7, and
nontumorigenic breast epithelial MCF-10A cells were obtained
from American Type Culture Collection (Manassas, VA). BC
cells were maintained in DMEM/F12 supplemented with 10%
heat-inactivated fetal bovine serum (FBS). MCF-10A cells were
supplemented with 100 ng/ml cholera enterotoxin, 10 µg/ml
insulin, 0.5 µg/ml hydrocortisol, 20 ng/ml epidermal growth
factor, 5% horse serum, 10 mM HEPES (RPMI), and 2.2 g/L
sodium bicarbonate, while MCF-7 cells were supplemented with
0.5 nmol/L estradiol and 5 µmol/L insulin. All cultures were
maintained in growth medium in the presence of 100 units/ml
penicillin and 100 µg/ml streptomycin at 37C in a humidified
atmosphere of 5% CO2in air as a monolayer culture in plastic
culture plates. Growth medium was changed every 48 h, and
cells were passed when they reached 85–95% confluence, as
observed by light microscopy.
Cell Viability Assay (MTT Assay)
Cells were seeded in 96-well tissue culture plates (8.0 ×
103cells/well) and incubated with GFE for 24–96 h. After incu-
bation, the growth medium was removed and cells were washed
with HBSS and incubated for 4 h with MTT reagent solution.
The color intensity of the formazan solution, which reflects the
number of cells under the specific growth conditions, was mea-
sured at 570 nm using a Victor3multilabel plate reader (Perkin
Elmer, Waltham, MA). The cell density of treatment groups was
expressed as percentage of the control.
Caspase-3 Activity
Following GFE treatments, MDA-MB-468 cells were har-
vested in cell lysis buffer and incubated on ice for 1 h. Af-
ter centrifugation at 11,000 ×gfor 30 min, the supernatants
were collected and protein concentration and caspase-3 activ-
ity immediately measured using a detection kit following the
manufacturer’s protocol (TruPoint Caspase-3 Assay Kit, Perkin
Elmer Life and Analytical Sciences, Norton, OH).
Cell Cycle Analysis
MDA-MB-468 cells were treated by 0, 5, 25, 50, or
100 µg/ml GFE for 48 h. After trypsinization, cells were col-
lected, washed, and suspended in 0.5 ml PBS (1 ×106cells/ml).
The cell suspension was added to 4.5 ml of 70% ethanol and
stored at 4C for 2 h. After centrifugation, cells were washed
with PBS and resuspended in 1 ml PI/ Triton X-100 staining
solution with DNase-free RNase A at 37C for 30 min before
analysis by the FACS Aria flow cytometer (BD Bioscience, San
Jose, CA). The experiments were conducted in triplicate.
Analysis of EGFR mRNA Expression Using qRT-PCR
After collecting 1.5 ×106of MDA-MB-468 cells which
were treated by 5 µg/ml (low) and 100 µg/ml (high) GFE
for 48 h, total RNA was isolated using the RNeasy Mini Kit
(Qiagen Company, Valencia, CA) and quantified by UV ab-
sorbance. cDNA was generated using 10 ng of RNA and iScript
Reverse Transcription Reagents as described in the manufac-
turer’s protocol (Bio-Rad). Primers were designed using Beacon
Designer 5 (Premier Biosoft International, Palo Alto, CA). The
oligonucleotide primers specific for EGFR and GAPDH (EGFR:
forward primer, 5-CCGTCGCTATCAAGG AATTAAG-3;re-
verse primer, 5-GTGGAGGTGAGGC AGATGG-3; GAPDH:
forward primer, 5-TTGGTATCGTGGAAGGACTC-3; reverse
primer, 5-TAGAGGCAGGGATGATGTTC-3) were used.
PCR and analysis of PCR products were performed using the
iCycler (Bio-Rad) and the SYBR-green detection system. Data
were analyzed using a comparative threshold cycle (Ct) method.
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GRAVIOLA FRUIT EXTRACT AND BREAST CANCER 797
Each sample was run in triplicate in separate tubes to permit
quantification of target genes normalized to controls, GAPDH.
Athymic Nude Mice
Five-wk-old female athymic BALB/c (nude) mice were pur-
chased from Charles River Laboratories (Wilmington, MA).
During the study, the mice were maintained under the standard
light/dark cycle (12-h light, 12-h dark).
Animal Treatment and Analysis of Tumor Growth
After 1 wk of acclimatization, MDA-MB-468 cells [1 ×
105cells/40 µl of Matrigel (Collaborative Biomedical Products,
Bedford, MA), 4 spots/mouse] were injected into the back of
the athymic mice. Mice were randomly divided into 2 groups,
MDA-MB-468 control and 200 mg GFE/kg diet (n=6), and
dietary treatment began. The selected daily GFE dose was equiv-
alent to 500–700 g of fresh fruit (2–3 fruits), reflecting rational
daily human consumption levels. American Institute of Nutri-
tion 93 growth diet (AIN93G) semipurified diet (Dyets, Bethle-
hem, PA) was selected as a base diet for control mice as it has
been established that it meets all the nutritional requirements of
mice (15). Treatment animals were fed AIN93G diet plus GFE
(200 mg/kg diet) for 5 wk until the average tumor surface area
of the control group reached 90.8 mm2. Tumor surface area and
body weight were measured weekly, and surface area was deter-
mined using the formula [length/2 ×width/2 ×π] (16). Food
intake was measured throughout the study. At termination, tu-
mor wet weight was measured. Tumors and blood samples were
collected for further analysis. Animal husbandry, care, and ex-
perimental procedures were conducted in compliance with the
Principles of Laboratory Animal Care NIH guidelines, as ap-
proved by the Institutional Animal Care and Use Committee
(IACUC) at Virginia Tech (06–010-HNFE).
Western Blotting Analysis
The dietary GFE-induced changes in protein expression of
EGFR, phosphorylated-ERK (p-ERK), and p-Akt were exam-
ined. Blots were stripped and reprobed with antibody that rec-
ognizes total ERK or Akt. Frozen tumors (4 tumors/control
and 6 tumors/GFE) were pulverized with mortar and pestle in
liquid N2and lysed and homogenized in radioimmunoprecip-
itation assay (RIPA) buffer. Homogenates were centrifuged at
10,000 ×gfor 10 min at 4C and supernatant was collected
for analysis. Protein from tumors (10–15 µg) was loaded into
a 7.5% gradient gel, electrophoresed at 100 V for 1.5 h, and
transferred to nitrocellulose membrane at 100 V for 1 h. The
membrane was blocked for 1 h in 5% milk-TBST at room tem-
perature, washed 3×with TBST, incubated overnight at 4Cin
primary antibody (EGFR, p-ERK, and p-Akt, Santa Cruz; Santa
Cruz, CA) washed 3×with TBST, incubated for 1 h at RT
in secondary antibody (goat antirabbit-HRP, bovine antimouse-
HRP, goat antirabbit-HRP, respectively) (Santa Cruz Biotech-
nology, Santa Cruz, CA), and activated with SuperSignal West
Femto Maxiumum Sensitivity Substrate (Thermo Fisher Scien-
tific, Inc., Rockford, IL). The membrane was then exposed to
film and developed manually. Protein bands were analyzed us-
ing Image J (NIH, Bethesda, MD), and the bands from each film
were normalized to β-actin (1Ab goat, 2Ab bovine antigoat-
HRP).
Statistical Analyses
Values are expressed as the mean ±SEM. Data from in vitro
experiments, tumor wet weight, Western blot analysis, body
weight gain, and food intake were analyzed using 1-way or
repeated-measures analysis of variance according to the charac-
teristics of the data set using the SAS program (SAS, Cary, NC).
If the overall treatment F-ratio was significant (P<0.05), the
differences between treatment means were tested with Tukey’s
multiple comparison test. Differences were considered to be
significant if P<0.05.
RESULTS
GFE Treatment Selectively Inhibited Growth of Human
BC Cells
The inhibitory effect of GFE treatments (96 h) on the growth
of BC cells and nontumorigenic breast cells is shown in Fig. 1.
Among the BC cells, MDA-MB-468 cells were extremely sen-
sitive to GFE treatment; the cell growth was inhibited by 29.5%,
with the GFE concentration as low as 0.5 µg/ml, an effect that
was highly dose-dependent. The IC50 of GFE for MDA-MB-
468 cells was 4.8 µg/ml. In contrast, other BC cells, such as
MDA-MB-231 and MCF-7 cells, were less sensitive to GFE
treatments. GFE treatments did not affect the growth of nontu-
morigenic MCF-10A cells, even with a concentration as high
as 200 µg/ml (Fig. 1A). These observations suggest that GFE-
induced growth inhibition is not only cancer-specific but also
highly selective against the different BC cells. Therefore, MDA-
MB-468 cells were selected for further GFE investigation.
Time-Dependence of GFE Effect on the Growth
of MDA-MB-468 Cells
As shown in Fig. 1B, the time-dependence of GFE effect on
the growth of MDA-MB-468 cells was more significant when
the cells were treated with the lower concentrations of GFE
(1 and 2 µg/ml). When the BC cells were treated with the
higher concentrations of GFE (5–25 µg/ml), the cell growth was
predominately inhibited in the first 24-h incubation; the relative
cell density remained stable when time was extended to 96 h.
In Fig. 1C, the cell density of MDA-MB-468 was remarkably
decreased, and numerous cells shrank after 48-h treatment with
25 µg/ml GFE.
GFE Treatment Induced Apoptosis in MDA-MB-468
BC Cells
The extent of apoptosis was determined by measuring the
level of caspase-3 activation following GFE exposure (Fig. 2A).
GFE treatment activated the caspase-3 activity in a dose-
dependent manner. The enzyme activity was increased by 17.8%
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798 Y. DA I E T A L .
FIG. 1. Graviola fruit extract (GFE) treatment selectively inhibited the growth
of human BC cells. A: Dose-response of GFE on human BC cells (MDA-
MB-231, MDA-MB-468, and MCF-7) and nontumorigenic MCF-10A cells.
Cells were grown in experimental growth medium of GFE for 96 h (n=3).
B: Time- and dose-effects of GFE on MDA-MB-468 cells. Cells were grown in
growth medium containing GFE for durations indicated. Data are expressed as
the percentage of control cells, mean ±SEM (n=3). C: Representative image
of MDA-MB-468 cells 48 h after treatment with GFE (25 µg/mL) or vehicle.
Cells were seeded at 5 ×104cells/mL on a 48-well plate for 48 h with the GFE
treatment of 0 and 25 µg/mL (n=3).
and 91.9% after treatment with GFE at 12.5 and 200 µg/ml me-
dia, respectively (P<0.05).
GFE Treatment Induced G1 Phase Arrest in MDA-MB-468
BC Cells
To further elucidate the mechanisms of GFE-induced growth
inhibition that may accompany the apoptosis, we examined the
effect of GFE treatment on cell cycle distribution. Concomitant
with the growth inhibitory effect, GFE induced G0/G1-phase
arrest in a dose-dependent manner (Fig. 2B and 2C). The G0/G1
population was increased by 3%, 5%, and 6% after 24-h GFE
treatments at 25, 50, 100 µg/ml, respectively. This increase
in the G0/G1 population paralleled a concomitant decrease in
the S and G2/M populations. Concurrently, the apoptotic cell
population of MDA-MB-468 cells was significantly increased
after 24-h GFE treatment. The G1/S ratio has been used as an
index of G1 arrest (17). The G1/S ratios of the GFE-treated
cells were significantly higher than the control (P<0.05) and a
constant increasing pattern of the G1/S ratio was observed with
increasing GFE concentrations.
GFE Treatment Downregulated mRNA Expression
of EGFR in MDA-MB-468 Cells
MDA-MB-468 cells contain an amplified EGFR gene, and
consequently these cells show very high expression levels of
EGFR (1.5 ×106receptor molecules/cell) (8). Previous experi-
ments have shown that MDA-MB-468 cells were most sensitive
to GFE treatment, followed by MDA-MB-231 and MCF-7 cells.
Western blot was conducted to compare EGFR protein levels
in these cell lines. Ten µg of total protein were separated by
7.5% SDS-PAGE and transferred to nitrocellulose membranes.
The proteins were probed with a rabbit anti-EGFR polyclonal
antibody. The ratio of EGFR/β-actin was 4.76 ±0.62 for MDA-
MB-468, 0.58 ±0.09 for MDA-MB-231, and 0.02 ±0.01 for
MCF-7. The result indicated that the selective GFE-induced
growth inhibition of MDA-MB-468 cells may be associated
with the inhibition of EGFR signaling. Using qRT-PCR to mea-
sure EGFR mRNA level, it was observed that GFE treatment at
5 and 100 µg/ml significantly downregulated the EGFR gene
expression by 30 ±3.5% and 54 ±8.4%, respectively (P<
0.05).
Dietary GFE Treatment Significantly Inhibited the Growth
of MDA-MB-468 Tumors Implanted in Athymic Mice
The promising in vitro results prompted us to further ex-
amine, via a dietary intervention experiment, whether GFE ex-
erts similar anticancer effects in vivo. After 5 wk of dietary
GFE treatment, average tumor surface areas were 90.82 ±9.15
mm2in the control group and 80.97 ±5.62 mm2in the GFE
(200 mg/kg diet) group, which is not a significant difference.
However, we observed a significant reduction of tumor wet
weight in the GFE group (reduced by 32%). Average tumor
weight was 253.3 ±54.7 mg in the control group, and 171.2
±23.2 mg in the GFE group (P<0.05). There was no sig-
nificant difference in food intake between the control and the
GFE treatment group (data not shown). However, there was a
significant difference in body weight gain between the 2 groups.
Body weight gain was calculated using the following formula:
body weight gain =(body weight at Week 5 – tumor weight) –
(body weight at Week 1). Average body weight gain was 2.6 ±
0.4 g for the control group, and 0.5 ±0.4 g for the GFE group,
respectively (P<0.05).
Dietary GFE Treatment Significantly Reduced the Protein
Expression of EGFR, p-ERK, and p-EGFR in Tumors
As shown in Figs. 3 and 4, dietary GFE treatment inhib-
ited the protein expression of EGFR, p-ERK, and p-EGFR in
MDA-MB-468 tumors by 56%, 54%, and 33%, respectively
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GRAVIOLA FRUIT EXTRACT AND BREAST CANCER 799
FIG. 2. Graviola fruit extract (GFE) treatment induced apoptosis in MDA-MB-468 BC cells. A: Caspase-3 activity as a function of different concentrations
of GFE. Cells were treated with various concentrations of GFE for 48 h and extracted. Data are expressed as a percentage of control cells, mean ±SEM (n=
3), and asterisks indicate a statistically significant difference compared to control cells (P<0.05). B and C: Cell cycle distribution after exposure to different
concentrations of GFE. Cells were grown in experimental media containing 0, 25, 50, or 200 µg/ml of GFE for 24 h. Data are expressed as a percentage of the
total population of cells receiving the respective treatments, mean ±SEM (n=3). Asterisks indicated a statistically significant difference compared to control
cells (P<0.05).
(P<0.05). However, no change was detected in the protein
expression of p-Akt in tumors.
DISCUSSION
Identification of novel, targeted chemotherapeutics that can
selectively inhibit tumors is of major importance in efforts to re-
duce the burden of BC. One such strategy to control BC growth
and metastasis could be its prevention and treatment by phyto-
chemicals, present in diets that specifically target BC cells. GFE
might be such a novel dietary agent that was initially identified
in our laboratory with anticancer potential through our prelimi-
nary screening of hundreds of food extracts and compounds on
growth inhibition of BC cells. GFE showed unusual selective
cytotoxicity to specific types of BC cells, suggesting that GFE
may selectively target specific mechanisms in certain BC cells
such as MDA-MB-468 cells.
To reveal the potential mechanisms governing the GFE-
induced growth inhibition of MDA-MB-468 cells, we first ex-
amined whether GFE induced apoptosis and/or cell cycle arrest.
Apoptosis regulates tissue homeostasis and is a critical mech-
anism for cancer chemoprevention and chemotherapy (18). In
BC, cells become resistant to apoptosis partially due to disrup-
tions of apoptotic signaling pathways and changes in the expres-
sion of enzymes associated with tumor resistance (19). In this
study, we found that GFE treatment induced a dose-dependent
increase in caspase-3 activity (Fig. 2A) and apoptotic cell death.
Cell cycle deregulation is closely associated with apoptosis,
and disruption of the cell cycle may eventually lead to apoptotic
death. Thus, inhibition of deregulated cell cycle progression
in cancer cells is another effective strategy to control tumor
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FIG. 3. Dietary graviola fruit extract (GFE) inhibited EGFR and p-ERK pro-
tein expression in tumors. Tumors in the control group (A, B, C) and GFE group
(D, E, F) were analyzed using Western blot analysis (4 and 6 tumors from the
control and GFE groups, respectively). Ratio of the target protein to the standard
protein expression level (bars ±SEM) is displayed on the Y axis. β-actin was
used as a standard for the quantity analysis. Asterisks indicated a statistically
significant difference compared to control cells (P<0.05).
growth (20). Chemotherapeutic agents often induce cell cycle
arrest at the G0/G1 or G2/M phases (17,21). Our data showed
that the increase of G0/G1 phase cells was accompanied by
a significant decrease of S-phase cells and moderate decrease
of G2/M phase cells, indicating that GFE treatment brought
about a blockage effect at the G1/S transition and induced dose-
dependent G0/G1 cell cycle arrest (Fig. 2B). These observations
suggest that GFE-induced growth inhibition of MDA-MB-468
cells is partly attributable to its induction of apoptosis and G0/G1
cell cycle arrest. However, the specific mechanism of the GFE
treatment on the cell cycle machinery and expression of several
related proteins deserves further investigation.
MDA-MB-468 cells exhibit EGFR gene amplification (23).
The extreme sensitivity of MDA-MB-468 cells to GFE treatment
prompted us to examine whether GFE can target EGFR signal-
ing. Indeed, we found that incubating MDA-MB-468 cells with
GFE resulted in significant downregulation of EGFR mRNA
expression in both lower (5 µg/ml) and higher concentrations
(100 µg/ml) of GFE. Molecular inhibition of EGFR signaling
has been shown to induce apoptosis and block cell cycle pro-
gression in cancer cells (9). GFE may also be acting in this
FIG. 4. Dietary graviola fruit extract (GFE) inhibited p-EGFR protein ex-
pression in tumors. Tumors were analyzed using Western blot analysis (4 and
6 tumors from the control and GFE groups, respectively). Numbers on the Y
axis represent the ratio of the target protein to the standard protein expression
level (bars ±SEM). β-actin was used as a standard for the quantity analysis. As-
terisks indicated a statistically significant difference compared to control cells
(P<0.05).
manner against MDA-MB-468 cells. We also noted that there
was no significant difference in tumor surface area between the
control and GFE groups during the feeding study. It is possi-
ble that tumor volume and mass are not directly proportional
because tumor growth is three-dimensional, and tumors consist
of tumor cells, inflammatory cells, edema, fibrosis, or necrosis.
Therefore, tumor wet weight at the termination would provide
a more accurate endpoint compared to surface area.
Activation of EGFR occurs frequently in both benign and
malignant hyperproliferative BC and triggers a cascade of down-
stream intracellular signaling pathways that contribute to tu-
morigenesis (9,24–26). This activation involves at least two ma-
jor pathways: the highly conserved Ras/mitogen activated pro-
tein kinase (MAPK)-dependent pathway [Ras/Raf/extracellular
signal-regulated kinase (MEK)/extracellular signal-regulated
kinase (ERK)] and the phosphatidylinositol 3-kinase (PI3-K)-
dependent pathway [PI3K/phosphatase and v-akt murine thy-
moma viral oncogene homolog (Akt)/mammalian target of ra-
pamycin (mTOR)]. The activation of the Ras/Raf/MEK/ERK
pathway results in cell survival, proliferation, migration, angio-
genesis, and inhibition of apoptosis (24). We examined whether
the GFE-induced tumor growth inhibition was attributable to
modulation of EGFR and these 2 major signaling pathways,
through measuring the protein expression of EGFR, p-EGFR,
p-ERK, and p-Akt in tumors. Dietary GFE, at a rational human
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GRAVIOLA FRUIT EXTRACT AND BREAST CANCER 801
exposure level (200 mg/kg diet), significantly inhibited pro-
tein expression of EGFR and p-EGFR and phosphorylation
of ERK but not of Akt. These results suggested that di-
etary GFE treatment may downregulate EGFR expression
and Ras/Raf/MEK/ERK pathways in vivo resulting in tumor
inhibition.
In summary, this is, to the best of our knowledge, the first
report demonstrating that a dietary agent at rational human ex-
posure levels significantly downregulates EGFR expression and
inhibits the growth of EGFR-overexpressing human BC cells
both in vitro and in vivo. GFE selectively inhibited the growth
of MDA-MB-468 cells without any effect on nontumorigenic
cells, suggesting that GFE possesses selective antitumor proper-
ties. We further confirmed the anti-EGFR and antitumor activi-
ties of GFE in vivo. Dietary GFE significantly inhibited tumor
growth in nude mice xenografts, suggesting that GFE might be
developed as a novel mechanism-based dietary agent for the
prevention and treatment of specific human BC. Further investi-
gation of the above observed inclinations and active components
in GFE is essential to establish solid grounds for the possible
future utilization of this dietary extract as a chemopreventive
agent.
ACKNOWLEDGMENTS
This study was partly supported by the Thomas F. and Kate
Miller Jeffress Memorial Trust.
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