Keledek antikanser
Polyphenol-rich sweet potato greens extract inhibits proliferation and induces apoptosis in prostate cancer cells in vitro and in vivo
Prasanthi
Karna,† Sushma
R. Gundala,† Meenakshi V. Gupta,1
Shahab A. Shamsi,2
Ralphenia D. Pace,3
Clayton Yates,4
Satya Narayan,5
and Ritu
Aneja*
Abstract
Sweet
potato (Ipomoea batatas) leaves or greens, extensively consumed as a
vegetable in Africa and Asia, are an excellent source of dietary polyphenols
such as anthocyanins and phenolic acids. Here, we show that sweet potato greens
extract (SPGE) has the maximum polyphenol content compared with several
commercial vegetables including spinach. The polyphenol-rich SPGE exerts
significant antiproliferative activity in a panel of prostate cancer cell lines
while sparing normal prostate epithelial cells. Mechanistically, SPGE perturbed
cell cycle progression, reduced clonogenic survival, modulated cell cycle and
apoptosis regulatory molecules and induced apoptosis in human prostate cancer
PC-3 cells both in vitro and in
vivo. SPGE-induced apoptosis has a
mitochondrially mediated component, which was attenuated by pretreatment with
cyclosporin A. We also observed alterations of apoptosis regulatory molecules
such as inactivation of Bcl2, upregulation of BAX, cytochrome c release and
activation of downstream apoptotic signaling. SPGE caused DNA degradation as
evident by terminal deoxynucleotidyl transferase-mediated dUTP-nick-end
labeling (TUNEL) staining of increased concentration of 3′-DNA ends.
Furthermore, apoptotic induction was caspase dependent as shown by cleavage of
caspase substrate, poly (adenosine diphosphate-ribose) polymerase. Oral
administration of 400 mg/kg SPGE remarkably inhibited growth and progression of
prostate tumor xenografts by ∼69% in nude mice, as shown by tumor volume
measurements and non-invasive real-time bioluminescent imaging. Most
importantly, SPGE did not cause any detectable toxicity to rapidly dividing
normal tissues such as gut and bone marrow. This is the first report to
demonstrate the in vitro and in
vivo anticancer activity of sweet
potato greens in prostate cancer.
Introduction
Nearly
one-third of all cancer deaths in the USA can be prevented through appropriate
dietary modification (1–3).
Regular consumption of fruits and vegetables (five servings per day) (4)
is highly recommended today in the American and European diet, mainly because
the constituent phytochemicals, in particular, polyphenols, they contain are
known to play important roles in long-term health protection, notably by
reducing the risk of chronic and degenerative diseases including cancer (5,6).
Prostate cancer is particularly amenable to dietary chemopreventive strategies
since it presents a significantly large-window of latency (∼20–30 years) and its
mean age of diagnosis is ∼68
years (7–10).
About 35 plant-based foods identified by the NCI display effective anticancer
properties including garlic, turmeric, cruciferous vegetables (e.g. broccoli,
brussels sprouts, cabbage) and grape seed extracts (8,11–14).
Many fruit and vegetable whole extracts have also been tested for their
efficacy in inhibiting prostate cancer growth (7,8,10,13,15).
Plant
polyphenols, a class of naturally occurring water soluble phenolic compounds,
are crucial for optimal human health benefits and are being increasingly
recognized owing to their abundance in fruits, vegetables and derived
foodstuffs (16).
The conformational flexibility of polyphenols facilitates complex
oligo/polymeric assemblies that enable plants to take advantage of the
remarkably diverse range of biophysicochemical properties exhibited by the
phenol functional group thus making plant polyphenolics as unique and
intriguing natural products (16).
No wonder polyphenols have sparked a new appraisal of diverse plant-derived
foods and beverages such as tea, red wine, coffee, cider, chocolate as well as
many other food commodities derived from fruits, including berries. The ability
of phenolics to homolytically release a hydrogen atom is one of the fundamental
processes that underlie the acclaimed health-benefiting antioxidative property
of polyphenolics to act as scavengers of free radicals and reactive oxidative
species that may drive malignant transformation and carcinogenesis (16).
Sweet
potato (Ipomoea batatas) leaves or greens are commonly consumed as a
fresh vegetable in West Africa and Asia, in particular, Taiwan and China (17).
Rich in vitamin B, β-carotene, iron, calcium and zinc, sweet potato greens
(SPG) are highly nutritive and contain as many vitamins, minerals and other
nutrients as spinach (18). SPG are
an excellent source of antioxidative polyphenolics, namely anthocyanins and
phenolic acids such as caffeic, monocaffeoylquinic (chlorogenic),
dicaffeoylquinic and tricaffeoylquinic acids (19,20).
The major anthocyanins in SPG are cyanidin-type rather than peonidin-type (21).
The constituent polyphenolics of SPG display antimutagenic, antidiabetic,
antibacterial, anti-inflammatory and anticancer activity (18,22). The
chemopreventive action of SPG is suggested by a case–control study in Taiwan
reporting that higher SPG consumption is associated with reduced lung cancer
risk (23).
Although
sporadic studies have reported identification of bioactive polyphenolics and
anthocyanin constituents of SPG (24),
there has, heretofore, not been a study that offers a detailed evaluation of
the anticancer potential of sweet potato greens extract (SPGE). To the best of
our knowledge, we are the first to investigate the anticancer attributes of
SPGE in vitro and in vivo and to develop it as a
mechanism-based anticancer agent for prostate cancer. In this study, we examine
the anticancer effects of SPGE in a panel of prostate cancer cells by evaluating
its effects on cellular proliferation, cell cycle progression and apoptosis.
Our results demonstrate that SPGE causes growth inhibition by inducing a G1
phase arrest followed by a mitochondrially mediated caspase-dependent intrinsic
apoptosis in prostate cancer, PC-3 cells. In vivo studies show that
SPGE remarkably inhibits tumor growth of subcutaneously implanted PC-3 human
tumor xenografts in nude mice models without any detectable toxicity.
Materials and methods
Cell culture, antibodies and reagents
Human
prostate cancer cell lines (LNCaP, DU145, PC-3, C4-2, C4-2B) were cultured in
RPMI medium (Mediatech, Manassas, VA) with 10% fetal bovine serum.
Luciferase-expressing PC-3 cells (PC3-luc) were from Calipers (Hopkinton, MA)
and were maintained in modified Eagle's medium with 10% fetal bovine serum.
Antibodies to cyclin D1, cyclin A, cytochrome c, Bcl2, phospho-Bcl2, cleaved
caspase-3 and cleaved poly (adenosine diphosphate-ribose) polymerase (PARP)
were from Cell Signaling (Beverly, MA). BAX, p21, cyclin E, p53 and β-actin
were from Santa Cruz Biotechnology (Santa Cruz, CA). Chlorogenic acid (ChA) and
caffeic acid (CA) were from Sigma (St Louis, MO).
Preparation of SPGE and estimation of polyphenolics
Forty-five-day-old
sweet potato (I.batatas) greens [Whatley/Loretan, (TU-155) variety] were
obtained from Tuskegee University Agriculture Department. Extracts were
prepared by soaking shade-dried leaves in methanol overnight for three
consecutive days. The supernatant was collected daily and finally concentrated in vacuo
(Buchi-Rotavap) followed by freeze drying to powder using a lyophilizer. SPGE
stock solution was prepared by dissolving 200 mg/ml dimethyl sulfoxide and
various concentrations were obtained by appropriate dilutions. Batch-to-batch
variation was evaluated by analysis of total polyphenolic (∼6.5/100 g) (25,26)
and anthocyanin (∼10.8
Color value/g powder) (21)
contents, which was observed to be consistent across batches of similar age.
In vitro cell proliferation and colony survival assay
Cells
plated in 96-well format were treated with gradient concentrations (1–1000
μg/ml) of SPGE the next day. After 72h SPGE treatment, cell proliferation was
determined using the Alamar Blue assay. For the colony assay, PC-3 cells were
seeded at appropriate dilutions (∼100 cells per well) and were treated with 250 μg/ml
SPGE for 48 h, washed and replaced with regular RPMI-medium. A colony was
arbitrarily defined to consist of at least 50 cells. After 10 days, colonies
were fixed with 4% formaldehyde, stained with crystal violet and counted.
n vivo tumor growth, SPGE treatment and bioluminescent imaging
Six-week
old male nude mice were obtained from NCI (Frederick, MD) and 1 × 106
PC-3-luc cells in 100 μl phosphate-buffered saline were injected subcutaneously
in the right flank. When tumors were palpable, mice were randomly divided into
two groups of eight mice each. Control group received vehicle
(phosphate-buffered saline with 0.05% Tween-80, pH = 7.4) and the treatment
group received 400 mg/kg body wt SPGE daily by oral gavage. Tumor growth was
monitored in real time by bioluminescent imaging of luciferase activity in live
mice using the cryogenically cooled IVIS-imaging system (Calipers) with the
live imaging software. Briefly, mice were anesthetized with isoflurane, intraperitoneally
injected 25 mg/ml luciferin and imaged with a CCD camera. Integration of 20 s
with four binnings of 100 pixels was used for image acquisition and signal
intensity was quantitated as sum of all detected photon counts within the
lesion. Mice from vehicle or SPGE-treated groups were imaged twice a week
allowing temporal assessment of in
vivo tumor growth. All animal
experiments were performed in compliance with Institutional Animal Care and Use
Committee guidelines.
Histopathologic and immunohistochemical analyses
After
6 weeks of SPGE or vehicle feeding, mice were euthanized. Organs and tumors
were either formalin fixed or frozen immediately. Tumor or organ sections were
stained with hematoxylin and eosin. Cleaved caspase-3, cleaved PARP, Ki67 and
TUNEL staining of tumor sections was performed as described previously (29,30).
Microscopic evaluation was performed by a pathologist in a blinded manner.
Statistical analysis
The
mean and standard deviations were calculated for all quantitative experiments
using Microsoft-Excel software. The Student’s t-test was used to
determine the differences between groups with P-values of <0.05
considered as statistically significant.
Results
SPGE has the highest polyphenol content
Nature
has selectively enriched plants with the phenolic functional group as a special
means to equip and elaborate complex secondary metabolites useful for their
development and survival. It comes as no surprise that plant extracts, herbs
and spices rich in polyphenolics have been used for thousands of years in
traditional oriental medicine. Thus, we first attempted to determine the total
polyphenolic content of SPGE compared with several commercially available
vegetables like spinach, mustard greens, kale, okra, green onions and collard
greens (Figure
1). Estimating polyphenolic content in terms of ChA equivalents
expressed as milligram per liter, our data showed that SPG had the highest
polyphenolic concentrations which were ∼43% higher than spinach (Figure
1A). We also quantified the anthocyanin content and found that SPG
had ∼2.5-fold
higher anthocyanin pigments compared with spinach (Figure
1B). These data encouraged us to evaluate the antiproliferative
potential of SPGE that was investigated next.
SPGE is
highest in (A) polyphenolic content and (B) anthocyanin
content compared with other commercial vegetables like spinach, kale, okra,
mustard greens, collard greens and green onions. Values and error bars shown in
the graph represent average and standard deviations, respectively, of three
independent experiments (P < 0.05).
SPGE inhibits proliferation of human prostate cancer cells
Given
that prostate cancer has a long latency time and is ideal for chemopreventive
intervention by non-toxic dietary extracts, we asked if SPGE inhibited growth
of prostate cancer cells (LNCaP, DU145, PC-3, C4-2 and C4-2B) in a
concentration gradient-dependent manner. SPGE significantly inhibited cellular
proliferation of all prostate cancer cells with IC50 values in the
range of 145–315 μg/ml (Figure
2A). The order of sensitivity was
C4-2>LNCaP>DU145>C4-2B>PC-3, with C4-2 being the most sensitive and
PC-3 the least. Importantly, the IC50 of SPGE in normal prostate
epithelial cells (PrEC and RWPE-1) was between 1000 and 1250 μg/ml (Figure
2B), which was ∼5-fold higher than for cancer cells suggesting that
SPGE specifically targets cancer cells while sparing normal cells.
SPGE
inhibits the growth and reproductive capacity of prostate cancer cells. (A) Bar graphical
representation of IC50 values of SPGE for various prostate cancer
cells and (B) normal prostate epithelial cells. (C) Bar-graph representation
and photograph of crystal violet-stained surviving colonies from control and
SPGE-treated groups. (Di) Fluorescence micrographs of PC-3 cells stained for
Ki67 or 4′,6-diamidino-2-phenylindole. (Dii) Bar-graph quantitation of Ki67-positive or 4′,6-diamidino-2-phenylindole-stained
cells treated with vehicle or 250 μg/ml SPGE. Values and error bars represent
average and standard deviations, respectively, of three independent experiments
(P < 0.05).
Next, we
performed a clonogenic or colony formation assay that evaluates the capacity of
a cell to proliferate indefinitely upon drug removal to form a colony or clone (Figure
2C). The most resistant cell line (i.e. highest IC50),
PC-3, was selected for clonogenic assay and subsequent studies to delineate
mechanisms of SPGE action. While controls produced several colonies, only a
fraction of SPGE-treated cells retained the ability to form colonies. Figure
2C shows the effect of 250 μg/ml SPGE on the relative clonogenicity
of control and SPGE-treated PC-3 cells. Representative micrographs of colonies
in control and SPGE-treated cells shown at the apex of bar graphs quantitated
to a ∼80%
reduction in number and size of surviving colonies upon SPGE treatment (Figure
2C). We also examined nuclear Ki67 expression, which correlates well
with growth fraction and found that Ki67 immunostaining was significantly more
intense in control cells compared with 250 μg/ml SPGE-treated cells over 24 h (Figure
2Di). Bar graph quantitation of Ki67-positive cells scored in both
control and SPGE-treated cells showed a ∼82% decrease in treated cells (Figure
2Dii). Furthermore, 4′,6-diamidino-2-phenylindole staining (Figure
2Di) indicated a ∼5-fold increase in cells with nuclear fragmentation (Figure
2Dii) compared with controls, suggesting SPGE-induced apoptotic cell
death. In addition, trypan blue data showed that SPGE-induced cell death over
time (0, 12, 24, 48 and 72 h) at 250 μg/ml in PC-3 cells (Supplementary
Figure 1 is available at Carcinogenesis Online).
SPGE perturbs cell cycle progression and modulates cell cycle regulatory molecules
We
next asked if SPGE-mediated growth suppression was due to its cell cycle
intervention. To this end, we evaluated the effect of varying dose and time of
SPGE exposure on the cell cycle progression of PC-3 cells. Figure
3Ai and 3Aii show dose and time courses of SPGE treatment in a
three-dimensional format. SPGE caused cells to accumulate in the G1
phase at doses ≤100 μg/ml over 24 h and at a dose of 250 μg/ml up to 12 h (Supplementary
Figure 2 is available at Carcinogenesis Online). This was followed by a dose- and
time-dependent increase in sub-G1 population, representing cells
with hypodiploid (<2N) fragmented DNA, a hallmark of apoptosis. The
quantitation of sub-G1 population over varying dose levels and times
is shown in Figure
3Aiii and 3Aiv, respectively. Since SPGE arrested cell cycle in the
G1 phase at low doses and shorter time periods, we examined this
acute effect of SPGE on G1 phase regulators. Immunoblot analysis
revealed a decrease in protein levels of cyclin D1, cyclin A and cyclin E after
24 h of 250 μg/ml SPGE treatment (Figure
3B). An increase in Cip1/p21 levels was evident at 24 h, in
agreement with the G1 arrest (Figure
3B).
(A) SPGE perturbs
cell cycle progression by causing a G1-arrest and increases sub-G1
cell population, indicative of apoptosis. (Ai) Cell cycle
progression over dose (0–600 μg/ml) and (Aii) time (0–72 h) is shown in a three-dimensional
format. (Aiii) Bar graphs depicting sub-G1 population of
PC-3 cells treated with SPGE over varying doses and (Aiv) time. (*P
< 0.05 compared with controls). (B) Immunoblots of cell lysates treated in absence or
presence of 250 μg/ml SPGE for cyclins D1, A, E and p21. The protein levels
were determined by quantifying the pixel values of the protein bands using
ImageJ on the immunoblots and normalized to the measured value at 0 h
treatment. Uniform loading was confirmed by β-actin. (Ci) Confocal
micrographs of Annexin-V-positive cells (staining at cellular rim) upon SPGE
treatment for 48 h and (Cii) quantitation of Annexin-V- or TUNEL-positive cells
for vehicle and SPGE treatment over time determined flow cytometrically. (*P
< 0.05 compared with controls).
SPGE induces robust apoptosis
Although
an increase of sub-G1 population upon SPGE treatment indicated
fragmented DNA suggesting apoptosis, we validated apoptosis both qualitatively
and quantitatively by Annexin-V staining using confocal microscopy and flow
cytometry methods. Immunofluorescence confocal micrographs showed that SPGE
treatment for 48 h at 250 μg/ml externalized phosphatidylserine to the outer
leaflet of the plasma membrane (observed as a rim) in PC-3 cells, a hallmark of
early apoptosis (Figure
3Ci). Flow cytometric quantitation suggested a steady increase in
Annexin-positive cells to ∼37% at 72 h (Figure
3Cii). It is well appreciated that altered cellular morphology,
including membrane blebbing, formation of apoptotic bodies, disruption of
cytoskeleton, hypercondensation and fragmentation of chromatin characterize
termination of apoptosis. Thus, we next quantified the increase in
concentration of 3′-DNA ends due to DNA fragmentation using a flow
cytometry-based TUNEL assay. We found that SPGE-treated cells showed ∼42% TUNEL-positive cells
(Figure
3Cii) at 72 h compared with controls, suggesting extensive DNA
cleavage.
SPGE induces caspase-dependent apoptosis
Both
extrinsic and intrinsic apoptotic pathways are well recognized as major
mechanisms of cell death in most cellular systems (31).
Having identified that SPGE induced robust apoptosis, we next evaluated whether
the apoptosis was caspase driven. Our data showed that treatment of PC-3 cells
with SPGE did not result in caspase-8 activation and cleavage (data not shown)
indicating non-recruitment of the extrinsic apoptotic pathway. However, SPGE
demonstrated a strong time-dependent cleavage of caspase-3 and PARP, as
observed by immunofluorescence and immunoblotting methods (Figure
4Ai and Aii and Supplementary
Figure 3Ai
and Aii, available at Carcinogenesis Online). Caspase
involvement was further confirmed by measuring caspase-3/7 activity using a
fluorescent substrate (Figure
4Aiii). Activation of caspase-3/7 without an effect on caspase-8
suggested involvement of the intrinsic pathway. To establish that this was the
major mechanism of SPGE-induced apoptotic death in PC-3 cells, we pretreated
cells for 3 h with pancaspase inhibitor z-vad-fmk followed by a 48 h treatment
with 250 μg/ml SPGE treatment. The extent of apoptosis was then determined by
estimating the sub-G1 population flow cytometrically. We observed
that z-vad-fmk pretreatment significantly inhibited SPGE-induced apoptosis by ∼65% (P <
0.01), suggesting that cell death was primarily caspase mediated (Supplementary
Figure 3Bi–iii is available at Carcinogenesis Online).
SPGE
activates the intrinsic apoptotic pathway. (Ai)
Immunofluoresence micrographs of vehicle-treated controls and 250 μg/ml
SPGE-treated cells stained for cleaved caspase-3 and PARP. (Aii) Immunoblot
analysis of cleaved caspase-3 and cleaved PARP levels in cell lysates from
vehicle-treated controls and SPGE-treated PC-3 cells. (Aiii) Caspase-3/7
activity assay over time. SPGE alters mitochondrial transmembrane potential. (*P
< 0.05 compared with controls). (Bi) Histogram profiles and (Bii) quantitation
of cytosolic monomeric JC-1 in unstained, control and SPGE-treated cells that
were read flow cytometrically (*P < 0.05 compared with controls).
SPGE-induced collapse of transmembrane potential was measured by increased
green fluorescence indicated by a right shift in the fluorescence intensity
curve. (Biii) Immunoblot analyses for p-Bcl2, total Bcl2, BAX and
cytosolic cytochrome c. The protein levels were determined by quantifying the
pixel values of the protein bands using ImageJ on the immunoblots and
normalized to the measured value at 0 h treatment or controls.
SPGE induces mitochondrially mediated intrinsic apoptosis
We
further confirmed intrinsic apoptosis by measuring the collapse of
mitochondrial transmembrane potential (Ψm) and examining release of
mitochondrial cytochrome c into the cytosol (32).
The effect of 24 h SPGE treatment on Ψm was observed by staining
with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide
(JC-1), a cationic dye, which exhibits potential-dependent mitochondrial
accumulationm (33).
An increase in JC-1 monomeric form indicative of Ψm collapse was
quantitatively determined using flow cytometry. As seen in Figure
4Bi, 250 μg/ml SPGE-treated cells at 24 h showed a right shift in
the mean-fluorescence intensity of green JC-1 monomers compared with controls.
There was a ∼90%
increase in the mean-fluorescence intensity of SPGE-treated JC-1-stained cells
compared with controls (Figure
4Bii). Most often, disruption of Ψm accompanies
alterations in expression level of Bcl2 members, in particular, the ratio of
antiapoptotic Bcl2 to proapoptotic BAX. We found that a 24 h 250 μg/ml SPGE
treatment increased the levels of phosphorylated Bcl2 indicating its
inactivation, whereas total Bcl2 levels remained unchanged (Figure
4Biii). A significant increase in BAX levels was observed at 24 h of
SPGE treatment (Figure
4Biii). In addition, cytosolic cytochrome c was elevated upon a 24 h
SPGE exposure (Figure
4Biii). Thus, these data strongly indicated a mitochondrially driven
apoptosis upon SPGE treatment. We further confirmed the extent of contribution
of the mitochondrial pathway toward SPGE-induced apoptosis using cyclosporin A,
a mitochondrial permeability transition pore inhibitor. Our results show that
pretreatment of cells with cyclosporin A for 3 h before SPGE treatment for 24 h
resulted in ∼38%
sub-G1 population compared with ∼62% upon SPGE treatment alone (Supplementary
Figure 4A is available at Carcinogenesis Online). Our
experiments to study the drop in Ψm correlated with our flow
cytometry data, in that we observed a diminution of the number of cells with
depolarized mitochondria when cyclosporin A was added 3 h before SPGE treatment
compared with when SPGE was administered alone (Supplementary
Figure 4B is available at Carcinogenesis Online). This is
clearly indicative of the protective effect of cyclosporin A. These results
suggest that there is a significant mitochondrial component to the total
apoptotic response of SPGE.
Oral SPGE feeding significantly inhibits PC-3 tumor growth
Having
identified significant in vitro antiproliferative and proapoptotic
activity of SPGE, we were curious to examine the in vivo efficacy of
SPGE to inhibit human prostate tumor xenografts subcutaneously implanted in
athymic nude mice. We employed a PC-3 cell line stably expressing luciferase
(PC-3-luc) that allowed real-time visualization and monitoring of prostate
cancer growth non-invasively (34).
Animals in the treatment group were fed daily with 400 mg/kg body wt SPGE by
oral gavage for 6 weeks and treatment responses were followed by bioluminescent
imaging in longitudinal studies using the same cohorts of mice (Figure
5Ai). In vehicle-treated control animals, tumors showed unrestricted
progression (Figure
5Ai and Aii). In contrast, SPGE feeding showed a time-dependent
inhibition of tumor growth over >6 weeks (Figure
5Ai and Aii), though significant retardation was evident as early as
2–3 weeks post treatment (Figure
5Aii). Quantification of relative photon counts revealed a ∼69% reduction in tumor
volume with a confidence level of P <0.05 (n = 8, Figure
5Aii) at week 6 compared with vehicle-treated controls. To assess
overall general health and well being of animals during treatment, body weights
were recorded twice a week. SPGE treatment was well tolerated and mice
maintained normal weight gain (data not shown) with no signs of discomfort
during the treatment regimen. To corroborate our bioluminescent imaging data,
we also measured tumor volumes using a vernier caliper. As shown in Figure
5Bi, tumor volume measurements demonstrated that oral SPGE treatment
for 6 weeks (42 days) reduced tumor volume by ∼75%. All animals in the control group were euthanized
by day 42 postinoculation due to tumor overburden, in compliance with
Institutional Animal Care and Use Committee guidelines. At the end point of
animal experiments (week 6), the excised tumors (Figure
5Bii) were weighed and a ∼65% reduction in tumor weight was observed in
SPGE-treated group compared with controls. We next determined the longevity of
surviving mice by monitoring them for general health and well being for 10
weeks. Kaplan–Meier analysis revealed a significantly increased survival time
with 87.5% animals treated with SPGE surviving until 10 weeks (P <
0.05; Figure
5C). This was a significant prolongation of survival compared with
controls where median survival time was only 6 weeks.
Dietary
feeding of SPGE inhibits human prostate tumor xenograft growth in nude mice.
Male nude mice were subcutaneously injected with 106 PC-3-luc cells.
(Ai) Bioluminescent images indicating inhibition of tumor growth over a
period of time. (Aii) Graphical representation of the quantitative photon
count from control and SPGE-treated mice for 6 weeks. (Bi) Tumor growth
monitored (by vernier calipers) and presented as tumor volume in cubic
millimeters, over a period of 42 days. (Bii) Photographic images of excised tumors and graphical
representation of tumor weight. (C) Kaplan–Meier survival graphs of SPGE treatment over
10 weeks. (*P < 0.05, Aii, Bi).
In vivo mechanisms of SPGE-mediated reduction of tumor growth
To
evaluate the in vivo effect of SPGE feeding on the antiproliferative
response associated with tumor growth inhibition, tumor tissue lysates were
analyzed for cyclins (including cyclins D1, A, E) and cyclin-dependent kinase
inhibitor, p21, using immunoblotting methods (Figure
6A). SPGE treatment caused a decrease in cyclin D1, A and E, which
correlated with our in vitro findings in PC-3 cells (Figure
6A). In addition, p21 upregulation was evident as a potential
mechanism of cell cycle inhibition of tumor cells (Figure
6A), which was in accordance with the G1 phase cell cycle
arrest observed in vitro. In vivo apoptotic response of SPGE feeding in PC-3-luc tumor
xenografts was evaluated by caspase 3/7-activity assay and immunoblotting of
tumor lysates for cleaved caspase-3 expression. As expected, cleaved caspase-3
expression (Figure
6A) as well as caspase-3/7 activity (Figure
6B) was higher in SPGE-treated tumors compared with controls.
Tumor
tissue lysates express high apoptotic and low proliferation markers. (A) Western blot
analysis, (B) Caspase-3/7 activity (*P < 0.05), (Ci) hematoxylin
and eosin, (Cii) Ki67, (D) cleaved caspase-3 and PARP from control and 400
mg/kg body wt SPGE-treated mice tumors.
We further
asked if SPGE caused regression of xenografted tumors by inhibiting
proliferation and triggering apoptosis. Hematoxylin and eosin-stained tumor
sections from SPGE-treated animals revealed large areas of tumor cell death
seen as tumor necrosis adjacent to normal looking healthy cells. Significant
loss of tumorigenic cells in SPGE-treated animals (Figure
6Ci, right, arrow) was consistent with the therapeutic effect of
SPGE. However, some viable tumor cells were observed at the periphery of cell
death zones. In contrast, microsections from control tumor tissues revealed
sheets of tumor cells with high-grade pleomorphic nuclei and angiolymphatic
invasion (Figure
6Ci, left). Furthermore, Ki67-stained tumor sections from SPGE-fed
animals showed weak immunoreactivity (Figure
6Cii) compared with vehicle-fed animals. Tumor sections from
SPGE-treated groups also showed a marked increase in cleaved caspase-3 and PARP
staining (Figure
6D) compared with vehicle-fed controls, suggesting induction of
robust apoptosis in tumors from SPGE-treated mice.
Non-toxic effects of SPGE
Toxicity,
particularly in tissues with actively proliferating cells, remains a major
concern in prostate cancer patients treated either radiotherapeutically or by
chemotherapeutic drug regimes. We found that there were no detectable
differences in the histological appearance of tissues including the gut, liver,
spleen, lung, brain, heart, testes and bone marrow from vehicle and
SPGE-treated tumor-bearing mice (Supplementary
Figure 5 is available at Carcinogenesis Online). In
addition, colonic crypts from both mice groups showed comparable nuclear Ki67
staining (Supplementary
Figure 6 is available at Carcinogenesis Online), suggesting
that SPGE did not affect normal tissues with rapidly proliferating cells.
Furthermore, complete blood count (e.g. red blood cells, white blood cells,
lymphocytes, hemoglobin), serum biochemical-profile markers [alanine
transaminase, aspartate transaminase, alkaline phosphatase for hepatic function
and creatinine, blood urea nitrogen and electrolytes including potassium,
magnesium, sodium, calcium and chlorides for renal function] were within the
normal range and similar between the control and SPGE-treated groups (Supplementary
Figure 7 is available at Carcinogenesis Online).
Identification of bioactive phytochemicals in SPGE
Given
the significant activity of SPGE, we next attempted to examine and identify its
bioactive constituents. To this end, we first performed a simultaneous on-line
HPLC–UV and HPLC–mass spectrometry (MS) comparative detection in both positive
and negative ion modes for SPGE using acetonitrile (ACN):water (H2O)
solvent system (gradient conditions detailed in Supplementary
Figure 8 are available at Carcinogenesis Online). The
HPLC–UV chromatograms (Supplemenatry
Figure 8Ai and Bi is available at Carcinogenesis Online)
show the appearance of 11 peaks. However, when SPGE passed through the MS
detector after eluting from UV detector, new peaks 6a and 11a (Supplementary
Figure 8Aii and Aiii
is available at Carcinogenesis Online) and 9a, 10a, 10b and 12 (Supplementary
Figure 8Bii and Biii
is available at Carcinogenesis Online) appeared in both positive and
negative ion modes, which were lacking UV chromophores. Two bioconstituents,
ChA and caffeic acid (CA) with m/z values of 353.0 and 179.0, respectively,
have been successfully identified in SPGE (Supplementary
Figure 8C
is available at Carcinogenesis Online) using tandem-mass spectrometry
(MS–MS) technique. The multiple reaction monitoring comparison for the
respective product ions, 191 for ChA and 135 for CA, between SPGE (Supplementary
Figure 8Di is available at Carcinogenesis Online) and a
mixture of pure standards (Supplementary
Figure 8Dii
is available at Carcinogenesis Online) confirmed the presence of both
caffeic and ChAs in SPGE. However, two additional peaks (in boxes, Supplementary
Figure 8Di is available at Carcinogenesis Online) were
observed to be having the same m/z values as the product ion of ChA (which was
not seen in case of pure standards), thus raising a possibility of the presence
of ChA derivatives, which follow similar fragmentation pattern (353→191). Work
in our laboratory is underway to unravel the identity of the active ingredients
present in SPGE using state-of-art HPLC–MS techniques.
Discussion
The
management of advanced prostate cancer or prostate cancer after androgen
therapy failure poses a critical challenge because options such as radiotherapy
and chemotherapy are associated with serious side effects. Several studies in
recent years have convincingly shown that chemopreventive agents affect the
process of carcinogenesis by targeting pathways such as carcinogen activation,
detoxification, DNA repair, cell cycle progression, differentiation and
induction of apoptosis in transformed cells. Besides displaying potent
anticancer activity, the 'golden-rule' for an agent to qualify as a
chemopreventive is that it should be well-tolerated, non-toxic, easily
available and inexpensive.
Fruits
and vegetables are excellent sources of chemotherapeutic and chemopreventive
agents (35) and there is a uniformity
of opinion emphasizing consumption of five or more servings of fruits and
vegetables daily to minimize the risk of cancer (4).
Several plant-based food extracts have been shown to be effective in cancer
therapy and prevention such as ripe berry extracts and grape seed extracts (8,36–38).
Essentially, the beneficial effects of fruits and vegetables are due to their
constituent phytochemicals that include polyphenolics, anthocyanins,
carotenoids, alkaloids and nitrogen and sulfur compounds. These phytochemicals
have been shown to target multiple events of neoplastic stages to confer
therapeutic benefits and reduce overall cancer risk (39,40).
In addition, several reports indicate that a variety of naturally occurring
compounds such as grape seed extract, silibinin, green tea catechins and apples
also play an important role in the prevention and treatment of prostate cancer (41–44).
Although
widely consumed as a vegetable in several parts of the world such as West
Africa and Asia (17),
SPG represent an untapped food resource in the USA. According to a United
States Department of Agriculture report, the greens can be consumed in several
forms including raw, cooked, steamed and processed. In addition, the
polyphenolic content in leaves is much higher than in other parts of sweet
potato such as the petioles, outer skin and storage root (18).
Several other reasons exist that merit the encouragement of SPG as a more
common vegetable in the USA. Firstly, oxalic acid content, which is a concern
in vegetables because of its predisposition to form crystals within the kidneys
is roughly one-fifth in SPG compared with spinach. Secondly, as a crop, SPG is
more tolerant to diseases, pests and moisture than any other leafy vegetable
grown tropically. SPG may be grown even during monsoon season of the tropics
thus making it the only vegetable that can be grown right after floods or
typhoons. Finally, this vegetable can be harvested several times during the
year (24). Because of these
attributes, sweet potatoes one of the crops selected by US National Aeronautics
and Space Administration (NASA) to be grown in a controlled ecological life
support system as a primary food source (45).
Several
groups, mostly from Japan, have characterized various polyphenolics and
anthocyanins present in SPG (21).
A recent study reported the growth-suppressive activity of sweet potato leaves
in colon cancer cells (19).
Given the several health-promoting attributes of SPG, the principle objective
of the present study was to evaluate and establish the anticancer efficacy and
associated mechanisms of SPGE treatment in human prostate cancer cells in
vitro and to translate these findings to an in vivo preclinical
cancer model. Our study reveals that SPGE causes cell growth inhibition induces
G1 phase arrest accompanied by upregulation of p21 and induction of
apoptosis in PC-3 cells. In these studies, downregulation of cell cycle
effectors, in particular the G1 cyclins, including cyclins D1, A and E is
revealed as a plausible antiproliferative mechanism of SPGE in PC-3 cells.
Selective
induction of apoptosis is a highly desirable trait of ideal chemopreventive and
chemotherapeutic regimens. Our data showed that SPGE efficiently induces
apoptosis in PC-3 cells as determined by Annexin-V- and TUNEL-staining assays.
Insights into molecular mechanisms reveal that SPGE-induced apoptosis is
largely mitochondrially mediated and associated with the collapse of the
transmembrane potential which results in the expulsion of key apoptogenic
molecules such as cytochrome c from the mitochondria. Oral feeding of SPGE
remarkably inhibits tumor growth, which is accompanied by antiproliferative and
proapoptotic effects together with a decline in cyclin levels, increased
expression of p21 and activated caspase-3.
Although
dismaying, it is true that present day chemotherapeutic approaches for cancer
patients can be as deadly as the disease itself. Toxicity normally includes
myelosuppression, immunosuppression, cardiotoxicity and peripheral neuropathy.
To assess safety of SPGE, we evaluated hematologic and histopathological
toxicity and found no deviations in hematologic variables andorgan-associated
toxicities in treated mice compared with controls. In addition, the acid–base
and electrolyte balances in SPGE-treated animals were also normal compared with
controls. Finally, evidence for the potential usefulness of SPGE as a
chemopreventive agent in humans was postulated using a body surface area
normalization method (46).
Using calculations involving the effective in vivo dose (400 mg/kg)
data, the human equivalent dose was determined to be 30 mg/kg SPGE. For an average,
70 kg adult, this translates to an equivalent dosage of ∼2.1 g SPGE. Considering
these facts and the United States Department of Agriculture's Food Guide
Pyramid, the human equivalent dose can be obtained from ∼85 g, or about a
half-cup of raw greens, which can be easily incorporated in a normal daily
diet.
The
presence of polar acids eluting early (ChA and CA) with retention times <10
min (Supplementary
Figure 8Ai and Bi
are available at Carcinogenesis Online), and relatively non-polar
compounds eluting later (peaks with retention times >30 min, Supplementary
Figure 8Ai and Bi
available at Carcinogenesis Online), might facilitate fractionation of
SPGE into two fractions via HPLC–UV, using semipreparative higher diameter HPLC
columns (allowing higher sample loading) to further identify and characterize
the bioactive constituent(s) present in SPGE. In the light of a recent paradigm
shift which recognizes that the anticancer attributes of fruits and vegetables
are due to an additive or synergistic interplay of the complex phytochemical
mixtures in whole foods (47),
it is perhaps likely that the whole SPG extract works through complementary and
overlapping mechanisms to offer the most optimal benefits (47,48).
In this case, single bioactive constituents may show anticancer activity at
much higher doses that may be toxic, whereas a mixture of multiple compounds
may show enhanced activity at lower non-toxic doses.
In
conclusion, our current study is the first to identify the remarkable
anticancer activity of SPGE in prostate cancer. Our data generate compelling
evidence for further evaluation of SPG as a chemopreventive regimen for
prostate cancer. Currently, work in our laboratory is underway to identify and
characterize the bioactive constituent(s) of SPGE that either work alone or in
an additive or synergistic manner to offer the significant anticancer benefits.
Funding
This work was supported by grant R00CA131489 to R.A. from the National Cancer Institute, and start-up funds from Georgia State University.
Acknowledgments
Conflict
of Interest Statement: None
declared.
Glossary
Abbreviations
ChA
|
chlorogenic acid
|
JC-1
|
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine
iodide
|
MS
|
mass spectrometry
|
PARP
|
poly (adenosine diphosphate-ribose) polymerase
|
SPG
|
sweet potato green
|
SPGE
|
sweet potato greens extract
|
Articles from Carcinogenesis are provided here courtesy of Oxford University Press
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