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Journal of Cancer Therapy, 2012, 3, 534-545
doi:10.4236/jct.2012.35069 Published Online October 2012 (http://www.SciRP.org/journal/jct)
1
Effect of Rosehip (Rosa canina) Extracts on Human Brain
Tumor Cell Proliferation and Apoptosis
Patrice Cagle1, Ombeni Idassi1, Janelle Carpenter1, Radiah Minor2, Ipek Goktepe3, Patrick Martin1
1Department of Biology, North Carolina A & T State University, Greensboro, USA; 2Department of Animal Sciences, North Caro-
lina A & T State University, Greensboro, USA; 3Department of Family and Consumer Sciences, North Carolina A & T State Univer-
sity, Greensboro, USA.
Email: pmmartin@ncat.edu
Received August 3rd, 2012; revised September 5th, 2012; accepted September 17th, 2012
ABSTRACT
Rosehips are blossoms from the wild rose (Rosa canina) and are commonly used as an herbal remedy. Previous reports
have shown that extracts made from rosehip plants are able to reduce cell proliferation of cancer cells. In this study, we
investigated the efficacy of rosehip extracts in preventing cell proliferation of three human glioblastoma cell lines
A-172, U-251 MG and U-1242 MG cell lines. Each of the glioblastoma cell lines treated with rosehip extracts (1
mg/mL - 25 ng/mL) demonstrated a significant decrease in cell proliferation. The rosehip extract-mediated decrease in
cell proliferation was equal to or better than the decrease of cell proliferation observed when inhibitors of the MAPK
(U0126, 10 µM) or AKT (LY294002, 20 µM) signaling pathways were utilized. Additionally, pretreatment of the these
cell lines with Rosehip extracts (1 mg/mL - 25 ng/mL) selectively decreased AKT, MAPK, and p70S6K phosphoryla-
tion suggesting these extracts prevent glioblastoma multiforme cell proliferation by blocking both the MAPK and AKT
signaling mechanisms. Results from colorimetric cell death assays, cell cycle analysis by flow cytometry, as well as
western blot studies demonstrate that rosehip extracts inhibit cell proliferation but do not promote apoptosis. Moreover,
rosehip extracts were able to increase the efficacy of Temozolomide, a chemotherapeutic agent used to treat patients
with glioblastomas. Surprisingly, rosehip extracts demonstrated a greater inhibition of cell proliferation than in combi-
nation with Temozolomide (100 µM) or Temozolomide as a single agent. Taken together these data suggest that rosehip
extracts are capable of decreasing glioblastoma cell proliferation without promoting apoptosis and demonstrate a greater
cell proliferation inhibitory effect than Temozolomide. More importantly, rosehip extracts may serve as an alternative
or compliment to current chemotherapeutic regimens for glioblastomas.
Keywords: Rosehip; Rosa canina; Glioma; Glioblastoma Multiforme; Cell Proliferation; Apoptosis; Temozolomide;
MAPK; AKT
1. Introduction
In the past twenty years the incidence of primary brain
tumors has increased and as the population gets older this
trend is expected to continue. Glioblastoma multiforme
(GBM) is an aggressive neoplasm that is characterized
by an elevated, aberrant, proliferative capacity that is
accompanied by a diffuse pattern of brain invasion [1].
GBM is classified as a grade IV astrocytoma by the
World Health Organization (WHO) and is the most ma-
lignant form of adult brain tumor [2]. Due to the delete-
rious properties of GBMs they are associated with ex-
tremely high mortality rates. Despite recent advances in
chemotherapy, radiotherapy and surgical interventions,
the clinical outcome of GBMs is poor and a better under-
standing of glioma biology is needed in order to develop
better therapeutic options. Recent studies have investi-
gated the anti-oncogenic properties of natural products
and several of these studies have shown that natural
product extracts exhibit anti-oncogenic properties against
various types of cancer [3-7]. Specifically, there has been
a renewed effort to examine the anti-oncogenic proper-
ties of natural products for the treatment of GBM cells
[8-13]. However, the anti-oncogenic properties of rose-
hip extracts on GBM cells have not been widely investi-
gated.
Rosehip extracts (rosehips) are derived from the rose
plant (Rosa sp. Pl.) and can be derived from Rosa canina
(Dog rose, Rosaceae, rosehip) and Rosa villous [3,14].
Rosehip extracts have been studied extensively as anti-
inflammatory agents as well as an alternative treatment
for osteoarthritis [15-17]. Rosehip extracts have also
been shown to possess a large amount of phytochemicals
that include flavonoides and polyphenols that have the
potential to serve as antioxidants that prevent cell prolif-
Copyright © 2012 SciRes. JCT
Effect of Rosehip (Rosa canina) Extracts on Human Brain Tumor Cell Proliferation and Apoptosis 535
eration and migration in breast (MCF-7), colon (HT-29)
and cervical cancer (HeLa) cell lines [14,18,19]. How-
ever, neither rosehip extracts nor the bioactive compo-
nents of these extracts have been extensively studied for
their ability to prevent GBM cell proliferation.
AKT and MAPK signaling mechanisms are known to
contribute to the development and promotion of brain
tumors, especially gliomas. Genetic alterations resulting
in amplification of the epidermal growth factor receptor
(EGFR), p53 and phosphatase and tensin homolog
(PTEN) mutations have been associated with altered
AKT and MAPK signaling in GBMs [20]. AKT signal-
ing has been associated with increased cell proliferation,
invasion, angiogenesis and inhibition of apoptosis in
GBMs [21-25]. Silencing AKT with siRNA oligonucleo-
tides is able to block GBM cell proliferation and promote
apoptosis [26,27]. Reseveratrol in combination with AKT
silencing promotes GBM cell apoptosis [26]. Down re-
gulation of PI3K with siRNA, an upstream kinase for
AKT, also prevents GBM cell proliferation and promotes
apoptosis [28]. EGFR mutations and amplification are
activators of the MAPK pathway in GBMs and this
pathway has been shown to increase GBM cell prolifera-
tion, invasion and migration [20]. Recently, plant ex-
tracts have shown anti-tumor properties towards GBM,
however, these studies do not directly address the anti-
tumor capacity of rosehip extracts and also do not iden-
tify which signaling mechanism(s) are interrupted [18,
19].
In this study we investigated the efficacy of rosehip
extracts in three human glioblastoma cell lines to better
understand the role of rosehip extracts in preventing
glioblastoma cell proliferation. Additionally, we exam-
ined which cell signaling mechanisms were altered by
rosehip extracts and whether rosehip extracts promoted
apoptosis in glioblastoma cells. We show that rosehip
extracts are able to prevent cell proliferation through a
mechanism that involves inhibition of both the AKT and
MAPK signaling pathways, but does not promote apop-
tosis. Furthermore we find that rosehip extracts prevent
cell proliferation more effectively than Temozolomide, a
frequently used chemotherapeutic in the treatment of
glioblastoma.
2. Materials and Methods
2.1. Materials
Rosehip extracts were kind gifts from Dr. Ipek Goketpe
(North Carolina A & T State University). Delbucco’s
Minimal Essential Media and Minimal Essential Me-
dium-α (MEM-α) as well as the live/dead cell viability
assay were purchased from Invitrogen (USA). Fetal bo-
vine serum (FBS) used to maintain the cells were pur-
chased from Biowest (USA). 3-(4,5)-dimethlythiahiazo
(-z-yl0-3,5-diphenytetrazoliumromide (MTT) was pur-
chased from Calbiochem (USA). Temozolomide and
tubulin primary antibody were purchased from Sigma
(USA). U0126 and LY294002 were purchased from Cal-
biochem (USA). Antibodies against phosphorylated
MAPK (Tyr 202/204), phosphorylated AKT (ser473),
phosphorylated p70S6K (Thr421/Ser424), phosphory-
lated Rb (Ser807/811), total MAPK, Fra-1, AKT and
p70S6K were purchased from Cell Signaling Technolo-
gies (Massachusetts, USA). SpectraMax M5 (Molecular
Devices, USA) plate reader was used to collect MTT and
live/dead data. An Accuri (Becton Dickinson, USA)
Flow Cytometry was used to analyze cell cycle status.
2.2. Plant Material and Preparation of Crude
Extracts
Dried Rosehip calyces and crude Rosehip extracts were
supplied by Dr. Ipeke Goktepe (North Carolina A&T
State University). The freeze dried calyces were grounded
to powder and then stored at −20˚C. Rosehip powder (50
g) was serially extracted with 80% methanol. The mix-
ture of freeze dried powder and 80% aqueous methanol
were sonicated for 20 minutes with continual nitrogen
gas purging. The extracts were filtered through Whatman
No. 2 filter paper and evaporated to dryness under re-
duced pressure. The extracts were and stored at –20˚C.
The obtained residues of the crude extracts were tested
in human glioblastomas for cellular anti-proliferative
activity.
2.3. Cell Culture
The human GBM cell lines U-1242 MG, U-251 MG, and
A-172 were kindly supplied by Dr. Isa Hussaini (Univer-
sity of Virginia, and U-1242 MG) and Dr. Waldemar
Debinski (Wake Forest University, A-172 and U-251
MG). The U-251 MG and A-172 cells were maintained
in high glucose Dulbecco’s Modified Eagle’s Medium
(DMEM) (Invitrogen, USA) supplemented with 10%
FBS and 1% of penicillin/streptomycin (Fisher, USA).
The U-1242 cells were maintained in Minimum Essential
Media-α (MEM-α) supplemented with 10% FBS and 1%
penicillin/streptomycin. All cells were incubated at 37˚C
in a humidified atmosphere containing 5% CO2. Cultures
were maintained in 10 cm2 plastic dishes and serially
passaged following trypsinization using a TrypLE Ex-
press Stable Trypsin-Like Enzyme with Phenol Red so-
lution (Invitrogen, USA).
2.4. Cell Proliferation Assay
The effects of rosehip extracts on cell proliferation were
determined by MTT assays. Briefly, 104 cells were
seeded into a 96-well culture plate and incubated 4 - 6 h
for attachment. The cells were then treated with various
Copyright © 2012 SciRes. JCT
Effect of Rosehip (Rosa canina) Extracts on Human Brain Tumor Cell Proliferation and Apoptosis
536
concentrations of the rosehip extract (1 mg/mL, 250
µg/mL, 25 µg/mL and 25 ng/mL) for 48 h. The PI3K
(Phosphoinositide3-kinase) inhibitor LY294002 (20 µM)
and the MEK1/MEK2 inhibitor U0126 (10 µM) were ad-
ministered for 12 - 16 h (Calbiochem). Cells treated with
TMZ (100 µM) were treated for 24 h prior to MTT as-
says. To determine the time-course response of rosehip
extracts, cells were plated into 96-well plates, and rose-
hip was added to the culture medium and cell viability
was assessed with MTT assay at 48 h after the drug
treatment. Following rosehip exposure, 10 µL of MTT
reagent was added to each well of a 96-well assay plate
containing the cells in 100 µL of culture medium, and the
plates were incubated at 37˚C in 5% CO2 for 4 h. A
volume of 100 µL of a color development solution
(0.04N HCL/ Isopropanol) was added for 90 minutes,
after which the optical density at 570 nm was determined
using a Spectra Max M5 microplate reader. The optical
density was used to calculate proliferation rates after
exposure to the different concentrations of rosehip rela-
tive to that of control cells with no rosehip exposure. All
analyses were performed in triplicate, and the mean for
each experiment was calculated.
2.5. Protein Extraction and Western Blotting
Following exposure to rosehip extracts at the various
concentrations, the cells were washed with cold phos-
phate-buffered saline containing 0.2 mM sodium or-
thovanadate. The cells were then harvested and lysed on
ice with 125 µL of pre-cooled Triton lysis buffer con-
taining 1% Triton X-100, 0.2% Nonidet P-40, in the
presence of 2 mm EDTA, 2 mg/mL sodium orthova-
nadate, and 1x protease cocktail inhibitor. Cells were
centrifuged at 15,000 rpm for 1 min at 4˚C. The protein
concentration of the supernatant was determined by the
BCA assay (BioRad, USA) and equal amounts of protein
from control and drug-treated cells were boiled for 5 min
in SDS-PAGE buffer. Proteins were separated by
SDS-PAGE on 8% polyacrylamide gels and subse-
quently transferred onto PVDF membranes. For im-
munoblotting, PVDF membranes were incubated with
specific antibodies recognizing target proteins overnight
at 4˚C. The membranes were then incubated with HRP-
conjugated secondary antibody (1:2000) for 1 h at room
temperature and subsequently analyzed by enhanced
chemiluminescence (ECL) detection system (Thermo
Scientific, USA) and visualized by autoradiography. Tu-
bulin (1:5000) was used as loading controls.
2.6. Live-Dead Viability/Cytotoxicity Assay
The viability of the cell lines following extract treatment
was assessed using the LIVE-DEAD assay (Molecular
Probes, USA). The assay was performed according to the
manufacturer’s instructions to examine rosehip extract
cytotoxicity after 48 h. The assay utilizes two fluorescent
dyes to label live and dead cells. The cytoplasm of live
cells was stained with 2 μm Calcein AM (green) the nu-
cleus of dead cells stained with 4 μm EthD-1 (red).
Stained cells were viewed using the Olympus IX71 in-
verted fluorescent microscope. Images were captured
within 45 min of labeling using the Olympus DP 71
digital camera. The amount of dye in each cell was quan-
titatively measured using the SpectraMax M5 microplate
reader.
2.7. Cell Cycle Analysis by Flow Cytometry
Cells were plated in 6 cm plates and incubated under the
conditions described above. After 4 - 6 hr incubation, to
allow cell attachment, the cells were rinsed and incubated
in serum free media for 24 h. After 24 h, the media was
discarded and the cells were treated with various concen-
trations of the rosehip extract (250 µg/mL and 25 µg/mL)
for 48 hr. Staurosporine (1 µM) was administered for 18
h (Sigma, USA). After treatment, the cells were tryp-
sinized and pooled, then pelleted by centrifugation at 300
× g for 6 min. Cell pellets were washed with PBS, then
fixed in ice cold 70% ethanol, and stored at −20˚C for 2
hr. Fixed cells were washed with PBS followed by a 30
minute incubation at room temperature in the dark in a
staining solution consisting of PBS with 20 µg/mL
propidium iodide (Sigma) (Missouri, USA), 0.1% Triton
X-100 and 200 µg/mL RNase A (Sigma, USA). Stained
cells were analyzed on the Accuri C6 Flow Cytometer.
Using DNA histograms, FCS Express 4 software (Becton
Dickson, USA) was used to quantify cell cycle compart-
ments to estimate the percentage of cells distributed in
the different cell cycle phases. Each experiment was re-
peated 4 times and the results are indicative of 4 inde-
pendent studies.
2.8. Statistical Analysis
Experiments examining cell proliferation using MTT,
Live/Dead apoptotic analysis and cell cycle determina-
tion using flow cytometry were averaged and means and
s.e.m. from three individual experiments were analyzed
for statistical significance using a one-tailed ANOVA
and the Newman-Kuels multiple comparison test to de-
termine statistical significance.
3. Results
3.1. Rosehip Extracts Attenuate GBM Cell
Proliferation and Cell Cycle Progression
We determined the rate of GBM cell proliferation in vitro
by measuring the rate of MTT incorporated into three
human GBM cell lines, A-172, U-251 MG and U-1242
Copyright © 2012 SciRes. JCT
Effect of Rosehip (Rosa canina) Extracts on Human Brain Tumor Cell Proliferation and Apoptosis 537
MG cells following varying concentrations of rosehip
extracts (1 mg/mL, 250 µg/mL, 25 µg/mL and 25 ng/mL).
Each one of the human brain tumor cell lines assayed
demonstrated a significant decrease in cell proliferation
following treatment with various concentrations of rose-
hip extracts. Specifically, rosehip extract concentrations
ranging from 25 µg/mL to 1 mg/mL significantly de-
crease the rate of cell proliferation in A-172 cells (Figure
1(a)). U-251 MG and U-1242 MG cells demonstrated
higher sensitivity to rosehip extracts. In these cells the
rosehip concentrations ranging from 25 ng/mL to 1
mg/mL show a decrease of cell proliferation (Figures
1(b) and (c)). These data suggest that crude rosehip ex-
tracts decrease the rate of cell proliferation in human
brain tumor cell lines. The ability of rosehip extracts to
prevent cell proliferation was compared to the ability of
known PI3K/AKT (LY294002, 20 µM) and MAPK
(U0126, 10 µM) inhibitors. In each of the cell lines tested
the higher concentrations of rosehip extracts demon-
strated an equal or greater decrease of cell proliferation
than LY294002 and U0126 (Figures 1(a)-(c)). These
data suggest that rosehip extracts have a greater capacity
to prevent GBM cell proliferation than known inhibitors
of cell proliferation.
(a)
(b)
(c)
Effect of Rosehip Extract Concentration on the Rate of Cell
Proliferation in Human Brain Tumor Cells. A-172,
U-251MG and U-1242 MG cells were treated with rosehip
extracts in the presence of 10% FBS at the concentrations
indicatedin the figure and then incubated for 48 h. The rate
of cell proliferation were determined as described in the
materials and methods section. Each value is presented as
the mean sem of three independent determinations. The
columns in each graph are presented as relative values in
comparison to serum-starved cells. Bars labeled with dif-
ferent letters are significantly different from one another (P
< 0.05). Bars labeled with the same letter are not signifi-
cantly different (P > 0.05).
Figure 1. Crude rosehip extracts prevent brain tumor cell
proliferation.
3.2. Rosehip Extracts Diminish AKT and MAPK
Signaling Mechanisms
To investigate the intracellular mechanisms controlling
rosehip extract sensitivity observed in the brain tumor
cell lines, we examined the phosphorylation state of sev-
eral proteins. We first determined the inhibitory effect of
rosehip extracts on the phosphorylation levels of MAPK
(Erk1/2; Tyr 202/04) and the total protein levels of Fra-1,
a downstream target of MAPK and a regulator of GBM
formation and progression [29,30]. All cell lines were
serum starved for 18 h to reduce the basal phosphoryla-
tion levels, and then incubated in the presence of various
concentrations of rosehip extracts (1 mg/mL, 250 µg/mL,
25 µg/mL and 25 ng/mL), and 10% FBS. Additional
samples were serum starved as described above and in-
cubated in the presence of 10% FBS and either U0126
(10 µM) or LY294002 (20 µm), known chemical inhibit-
tors of the MAPK and AKT signaling pathways, respect-
tively. MAPK (Erk1/2; Tyr 202/04) phosphorylation in
A-172 (Figure 2(a)), U-251 MG (Figure 3(a)), and
U-1242 MG (Figure 4(a)) cells was decreased following
exposure to rosehip extracts and U0126, but not
LY294002. In addition to a decrease in MAPK (Erk1/2)
phosphorylation, Fra-1 (total) protein levels were also
decreased in response to rosehip treatment and U0126
treatment, but not LY294002. Previous studies have
shown that MAPK (Erk1/2) phosphorylation results in
Copyright © 2012 SciRes. JCT
Effect of Rosehip (Rosa canina) Extracts on Human Brain Tumor Cell Proliferation and Apoptosis
Copyright © 2012 SciRes. JCT
538
(a) (b)
Inhibitory Effect of Rosehip Extract Concentration on AKT andMAPK (Erk1/2) signalingin A-172 Cells. A-172 cells were treated with rosehip extracts inthe
presence of 10% FBS at the concentrations indicated in the figure and incubated for 48 h. (a) The phosphorylation status of MAPK and protein expression level
of Fra-1 was determined by a western blotting analysis; (b) The phosphorylation status of AKT and p70S6K was determined by a western blotting analy-
sis.Tubulin was used as a protein loading control.
Figure 2. In A-172 cells, a GBM cell line, rosehip extracts inhibit the MAPK and AKT signaling pathways.
(a) (b)
Inhibitory Effect of Rosehip Extract Concentrationon AKT andMAPK (Erk1/2) signaling in U-251MG Cells. U-251MG cells were treated with rosehip extracts
in the presence of 10% FBS at the concentrations indicated in the figure and incubated for 48 h. (a) The phosphorylation status of MAPK (Erk1/2) and protein
expression level of Fra-1 was determined by a western blotting analysis; (b)The phosphorylation status of AKT and p70S6K was determined by a western
blotting analysis. Tubulin was used as a protein loading control.
Figure 3. Rosehip extracts inhibit the MAPK and AKT signaling pathways In U-251 MG cells, a GBM cell line.
increased Fra-1 protein levels and increased transcript-
tional activity [31-33]. Taken together these data suggest
that rosehip extracts prevent MAPK (Erk1/2) phos-
phorylation and this inhibition of MAPK (Erk1/2) phos-
phorylation correlates with the reduction of cell prolif-
eration observed in A-172 cells (Figures 1(a)-(c)).
Additional studies investigated whether rosehip ex-
tracts could decrease the activity of the AKT cell prolif-
eration regulatory pathway. Each cell line was treated in
a similar manner as described above. Rosehip extracts
(all concentrations tested) prevent AKT phosphorylation
(ser 473) and p70S6K (Thr 421/Ser 424) phosphorylation
in A-172 cells, in a similar fashion as LY294002 (Figure
2(b)). AKT and p70S6K phosphorylation is only slightly
decreased by higher concentrations of rosehip extracts (1
mg/mL and 250 µg/mL) in U-251MG cells (Figure 2(b)).
Effect of Rosehip (Rosa canina) Extracts on Human Brain Tumor Cell Proliferation and Apoptosis 539
(a) (b)
Inhibitory Effect of Rosehip Extract Concentrationon AKT and MAPK (Erk1/2) signaling in U-1242MG Cells. U-1242MG cells were treated with rosehip
extracts in the presence of 10% FBS at the concentrations indicated in the figure and incubated for 48 h. (a) The phosphorylation status of MAPK (Erk1/2) and
protein expression level of Fra-1 was determined by a western blotting analysis; (b) The phosphorylation status of AKT and p70S6K was determined by a west-
ern blotting analysis. Tubulinwas used as a protein loading control.
Figure 4. Rosehip extracts inhibit the MAPK and AKT signaling pathways In U-1242 MG cells, a GBM cell line.
In U-1242 MG cells AKT and p70S6K phosphorylation
is decreased by rosehip extracts (Figure 4(b)). Specifi-
cally, rosehip extracts are able to decrease the phos-
phorylation of p70S6K at all concentrations tested in
U-1242 MG cells (Figure 4(b)), but only at the highest
tested concentrations in U-251 MG cells (Figure 3(b)).
These data indicate that rosehip can also prevent the ac-
tivation on the AKT cell signaling regulatory pathway in
A-172 and U-251MG cells (Figures 2(b) and 3(b)).
More importantly, these data indicate that rosehip ex-
tracts are able to inhibit multiple proliferation regulatory
mechanisms in the cell lines assayed.
3.3 Rosehip Extracts Do Not Promote Cell Death
In order to examine whether rosehip extracts were pro-
moting cell death in A-172, U-251 MG and U-1242 MG
cell lines, cells were cultured in the presence of 250
µg/mL rosehip extract and 10% FBS and the amount of
cell death was measured using a fluorescence colorimet-
ric analysis (as described in the materials and methods
section). The mean percentage of dead cells was deter-
mined following rosehip extract treatment and these val-
ues were compared to the values generated by A-172,
U-251 MG and U-1242 MG cells that were cultured in
10% FBS and staurosporine (1 µM) for either 3 h
(U-1242MG cells) or 24 h (A-172 and U-251MG cells)
in order to induce cell death. Since in cell proliferation
assays there was no significant difference observed be-
tween the 1 mg/mL and 250 µg/mL concentration of
rosehip extracts we used 250 µg/mL of rosehip extracts
to determine the apoptosis promoting capacity of the
extracts. Each cell line tested with rosehip extracts (250
µg/mL) did not promote cell death, but staurosporine
treatment did result in cell death (Figure 5). Figure 6
(these data correlate to the A-172 panel displayed in Fig-
ure 5) shows A-172 cells treated with staurosporine have
a much higher amount of cell death than cells treated
with rosehip extracts or cells cultured in serum contain-
ing medium without inhibitors (Figure 6, panels C, F, I).
These data suggests the prevention of cell proliferation
observed in cells treated with rosehip extracts is due to
inhibition/decrease of cell cycle progression and not cell
death.
Since rosehip extracts are able to prevent cell prolif-
eration in the cell lines tested, but do not promote cell
death we wanted to examine whether exposure to these
extracts resulted in PARP cleavage, a marker of apop-
tosis. To test further whether rosehip is promoting apop-
tosis, we used a western blot to assay for PARP cleavage.
PARP, a nuclear polymerase protein important for DNA
repair in response to stress, is cleaved from its 116 kDA
full size protein to a 89 kDA fragment, which in human
cells has been associated with cell apoptosis [34]. In
Figure 7 we show that rosehip extracts (all concentra-
tions) do not induce PARP cleavage in neither the A-172,
U-251MG nor U-1242MG cells (Figures 7(a)-(c)) when
compared to control cells either cultured under serum
starvation conditions or cultured in the presence of 10%
FBS. Additionally, cells cultured with FBS (10%) and in
the presence of U0126 and LY294002 do not display an
increased amount of cleaved PARP (Figures 7(a)-(c)).
However, A-172, U-251MG and U-1242MG cells treated
with staurosporine (1 µM) demonstrate a drastic increase
Copyright © 2012 SciRes. JCT
Effect of Rosehip (Rosa canina) Extracts on Human Brain Tumor Cell Proliferation and Apoptosis
540
Effect of Rosehip Extract Concentration on Promoting Cell Death. A-172, U-251 MG and U-1242 MG cells were
treated with rosehip extracts in the presence of 10% FBS at the concentrations indicated in the figure and incu-
bated for 48 h. Each cell line was also exposed to staurosporine (1 μM) for 24 h (A-172 and U-251 MG cells) or
3 h (U-1242 MG cells) to promote cell death. A live/dead colorimetric assay was used to determine the number
of dead cells. Mean percentages were calculated based on three independent experiments. The number in paren-
thesis represents the standard deviation. Values labeled with different letters are significantly different from one
another (P < 0.01). Values labeled with the same letter are not significantly different from one another (P > 0.05).
Figure 5. Rosehip extracts do not significantly increase the amount of cell death.
(a) (d) (g)
BF
(b) (e) (h)
Viable
cells
(c) (f)
(i)
Non-Viable
cells
+ Serum + RHP 250
μg
/mL + Stauros
p
orine 1
μ
M
Exposure to Rosehip extracts does not result in cell death. A-172 human GBM cell lines were treated with rose-
hip extracts for 48 h at 250 μg/mL. In addition to rosehip extracts, staurosporine (1 μM) was also used. The
amount of cell death was determined using a Live/Dead colorimetircassay.
Figure 6. Rosehip extracts do not promote cell death.
in the amount of cleaved PARP, demonstrating the affect
of apoptosis on the cleavage state of PARP in these cell
lines (Figures 7(a)-(c)). These data further emphasize
that rosehip extracts do not promote apoptosis in the
GBM cell lines assayed.
3.4. Rosehip Extracts Prevent Cell Cycle
Completion in Human GBM Cell Lines
Since GBM cells treated with rosehip extracts had de-
creased rates of proliferation and were not undergoing
Copyright © 2012 SciRes. JCT
Effect of Rosehip (Rosa canina) Extracts on Human Brain Tumor Cell Proliferation and Apoptosis 541
Effect of Rosehip Extract Concentration on PARP Cleavage in Human Brain Tumor Cells. A-172, U-251 MG and U-1242 MG cells were
treated with rosehip extracts in the presence of 10% FBS at the concentrations indicated in the figure and incubated for 48 h. Each cell line
was also exposed to staurosporine (1 µM) for 24 h (A-172 and U-251 MG cells) or 3 h (U-1242 MG cells) to promote cell death and PARP
cleavage. Cleaved PARP is represented by the lower band (89 kDA) and total PARP is represented by the upper band (116 kDA). (a) A-172
cells (b) U-251 MG cells and (c) U-1242 MG cells. Tubulin was used as a protein loading control.
Figure 7. GBM cells exposed to rosehip extracts do not demonstrate an increased amount of PARP cleavgage.
cell death we investigated the mechanism by which these
extracts were decreasing cell proliferation. Flow cytome-
try was used to analyze the cell cycle following exposure
to rosehip extracts. A-172 cells treated with rosehip ex-
tracts at a high concentration (250 µg/mL) and a low
concentration (25 µg/mL) show that cells exposed to
rosehip extracts are unable to maintain a normal cell cy-
cle (Figure 8). Specifically, rosehip extracts (both con-
centrations assayed) significantly prevented these cells
from exiting the G0/G1 phase of the cell cycle (Figure 8).
Cells treated with rosehip extracts have the same per-
centage of cells remaining in the G0/G1 phase of the cell
cycle as serum-starved cells (Figure 8).
Since rosehip extracts were able to slow cells from ex-
iting the G0/G1 phase of the cell cycle we determined the
level of retinoblastoma (Rb) tumor suppressor protein
phosphorylation (Ser 807/811) following exposure to
rosehip extracts (1 mg/mL, 250 µg/mL, 25 µg/mL and 25
ng/mL). Rb phosphorylation by a CDK inactivates tumor
suppressor (cell cycle inhibitory) function of Rb and
promotes continuation of the cell cycle [35]. A-172 cells
show a decrease of Rb phosphorylation at each concen-
tration of rosehip extracts, whereas control serum-cul-
tured (10% FBS) cells not exposed to rosehip extracts
cells do not (Figure 9). U-251MG cells exhibit a lowered
amount of Rb phosphorylation following exposure to
higher concentrations of rosehip extracts (1mg/mL and
G
0
/G
1
S G
2
/M
- Serum 88.20
b
1.86 6.21
b
+ Serum 64.75
a
9.43 20.50
a
Rosehip
(250 µg/mL)
88.83
b
1.29 4.17
b
Rosehip
(25 µg/mL)
81.55
b
4.49 10.28
b
Cell cycle modulation following exposure to rosehip extracts. A-172 cells
were treated with rosehip extracts in the presence of 10% FBS at the con-
centrations indicated in the figure and incubated for 48 h. Cells were then
fixed and stained with propidium iodide (1 mg/mL) to determine the per-
centage of cells in the representative portion of the cell cycle. The super-
script letters (a, b) denoted significant difference between percentage values
(a = P < 0.05); b = P < 0.01).
Figure 8. A-172 GBM cells treated with rosehip extracts
halt the cell cycle in the G0/G1 phase.
250 µg/mL); whereas U-1242MG cells only exhibited
substantial decrease in Rb phosphorylation when ex-
posed to the highest concentration of rosehip extracts (1
mg/mL) (Figure 9). These data demonstrate that rosehip
extracts are able to slow the rate of cell proliferation in
Copyright © 2012 SciRes. JCT
Effect of Rosehip (Rosa canina) Extracts on Human Brain Tumor Cell Proliferation and Apoptosis
542
Effect of Rosehip Extract Concentration on Retinoblastoma Protein Phos-
phorylation in Human Brain Tumor Cells. A-172, U-251 MG and U-1242
MG cells were treated with rosehip extracts in the presence of 10% FBS at
the concentrations indicated in the figure and incubated for 48 h. (top panel)
U-251 MG cells and (middle panel) U-1242 MG cells (bottom panel) A172
cells. Tubulin was used as a protein loading control.
Figure 9. Rosehip extracts decrease the phosphorylation
level of Retinoblastoma protein.
the GBM cell lines assayed by halting the cells in the
G0/G1 phase of the cell cycle via an Rb-mediated mecha-
nism.
3.5. Rosehip Extracts Have a Greater
Anti-Proliferative Capacity than a
Commonly Used Chemotherapeutic Agent
To better understand the chemotherapeutic potential of
rosehip extracts against human GBM cell lines, we tested
whether the antiproliferative capacity of rosehip extracts
compared to that of Temozolomide (TMZ). TMZ (also
known by the following brand names; Temodar, Temo-
dal and Temcad) is one of the current first line chemo-
therapeutic options used in the treatment of GBMs. TMZ
is an orally administered alkylating agent that can either
halt the cell cycle and/or initiate cell death [36]. Using
MTT assays we compared the rate of cell proliferation
following exposure to either rosehip extract alone (250
µg/mL), TMZ (100 µM) alone or a combination of the
rosehip extracts and TMZ [37]. We used rosehip extracts
at a concentration of 250 µg/mL since our previous cell
proliferation studies demonstrated that 250 µg/mL con-
centration of rosehip extracts significantly decreased cell
proliferation (Figures 1(a)-(c)). Surprisingly, in all of the
cell lines assayed, cells exposed to the rosehip extracts
(250 µg/mL) demonstrated a greater decrease in cell pro-
liferation when compared to cells exposed to TMZ (100
µM) (Figures 10(a)-(c)). These observed differences
were statistically significant (P < 0.05) in A-172 and
U-251 MG cell lines (Figures 10(a) and (b)). Addition-
ally, in U-251MG cells, TMZ (100 µM) demonstrated no
0.0
0.5
1.0
1.5
a
b
c
dd
Rel. Rate of Cell Proliferation
A-172
(a)
0.0
0.5
1.0
1.5
2.0
U-251 MG
Rel. Rate of Cell Proliferation
(b)
0.0
0.5
1.0
1.5
2.0
a
b
c
aa
Rel. Rate of Cell Proli feration
U-1242 MG
(c)
Effect of Rosehip Extract Concentration and Temozolomide on the Rate of
Cell Proliferation in Human Brain Tumor Cells. A-172, U-251 MG and
U-1242 MG cells were treated with rosehip extracts (250 µg/mL) in the
presence of 10% FBS and/or TMZ (100 µM) as indicated in the figure and
then incubated for 48 h. The rate of cell proliferation were determined as
described in the materials and methods section. Each value is presented as
the mean ± sem of three independent determinations. The columns in each
graph are presented as relative values in comparison to serum-starved cells.
Bars labeled with different letters are significantly different from one an-
other (P < 0.05). Bars labeled with the same letter are not significantly
different (P > 0.05).
Figure 10. Rosehip extracts and temozolomide prevent
brain tumor cell proliferation.
Copyright © 2012 SciRes. JCT
Effect of Rosehip (Rosa canina) Extracts on Human Brain Tumor Cell Proliferation and Apoptosis 543
inhibitory effect, whereas rosehip extracts (250 µg/mL)
significantly decrease the rate of cell proliferation (Fig-
ure 10(b)). These assays also show that the combination
of TMZ (100 µM) and rosehip extracts (250 µg/mL) do
not synergize to decrease cell proliferation more than
rosehip as a single agent (Figures 10(a)-(c)). These data
demonstrate that rosehip extracts have a greater capacity
to prevent in vitro GBM cell proliferation than a widely
used chemotherapeutic treatment of GBMs.
4. Conclusions
The anti-cancer effects of rosehip extracts have been
tested in various cancers; however, the cellular mecha-
nisms that are affected by rosehip extracts are not clear.
It has been suggested that the anti-cancer effects of rose-
hip extracts are related to the anti-oxidant and anti-pro-
liferative effects of these extracts [18,19]. The present
studies show that rosehip extracts prevent cell prolifera-
tion (Figure 1) and demonstrate the anti-proliferative
characteristic of rosehip extracts as seen in other cancer
cells [18,19]. Specifically these data show that rosehip
extracts are able to prevent cell proliferation in GBM
cells. The mechanistic studies presented in this study
show that the anti-proliferative effects of rosehip extracts
in GBM cells are associated with a decrease in AKT and
MAPK signaling, which are different signaling mecha-
nisms then observed in the anti-inflammatory and anti-
cancer effects in previous studies [18,19]. More impor-
tantly, neither of the aforementioned studies assayed the
anti-cancer effects of rosehip extracts on GBM cells nor
did these studies directly test which cellular mechanisms
were being negatively-regulated by the rosehip extracts.
[18,19]. Our mechanistic studies (Figures 2-4) demon-
strate a down regulation of Erk phosphorylation (Tyr
202/04) as well as Fra-1 protein levels a downstream
target of active Erk and a regulator of gliomagenesis
[29,30]. This inhibition shows that the MAPK cell sig-
naling pathway is negatively regulated by rosehip ex-
tracts and correlates with the decrease in cell prolifera-
tive capacity of these cells following exposure to the ex-
tracts. In addition to the MAPK kinase pathway the rose-
hip extracts also exhibit the ability to decrease AKT-
mediated cell signaling. Specifically, AKT phosphoryla-
tion (ser 473), which is associated with GBM cell prolif-
eration, survival and migration, is decreased when cells
are grown in the presence of rosehip extracts (Figures
2-4). Data presented also shows that p70S6K kinase
phosphorylation (Thr421/Ser424), which positively regu-
lates glioma cell proliferation and resistance to drug
therapy, is reduced after exposure to rosehip extracts
(Figures 2-4) [21,24]. Together these data are the first to
demonstrate that rosehip extract-mediated decrease of
GBM cell proliferation is regulated by the AKT and
MAPK cell regulatory mechanisms.
Tumbas and colleagues showed that a fraction of rose-
hip extracts containing quercetin was able to inhibit cell
proliferation in Hela (cervical cancer), MCF7 (breast
cancer) and HT-29 (colon cancer) cell lines [19].
Quercetin, a flavonoid, has been reported as one of the
active ingredients that can be elucidated from rosehip
extracts [12,13,38]. Purified quercetin has been tested
against U-87MG, U-251 MG and A-172 cells [38]. These
studies show that quercetin did not promote apoptosis
unless it was accompanied by TRAIL; however, cell pro-
liferation was not directly measured [38]. These data
correlate with our data demonstrating that rosehip ex-
tracts do not promote GBM cell apoptosis (Figures 5-7).
Specifically, in the cell lines tested, staurosporine was
the only agent that induced cell death and PARP cleav-
age, while none of the rosehip concentrations tested in-
creased the amount of cell death or PARP cleavage
(Figures 5-7). In another report, quercetin demonstrated
the ability to decrease U138MG, a human glioma cell
line, cell proliferation and induce cell death via an arrest
in the G2 checkpoint of the cell cycle [13]. Our result
with rosehip extracts demonstrate an arrest at the G0/G1
checkpoint in the cell lines tested (Figures 8 and 9). This
difference could be associated with the previously re-
ported use of purified quercetin versus a crude extract of
rosehip plants as described here [13]. It is possible that
the amount of quercetin in the extract is at lower concen-
tration levels and thus does not promote a G2 cell cycle
arrest. Alternatively, it is plausible that another compo-
nent of the extract may have a stronger arresting affect
on the G0/G1 cell cycle checkpoint. Future studies in the
laboratory will fractionate the crude rosehip extract and
determine which active ingredient(s) are regulating the
decrease in cell proliferation observed in our studies.
Importantly, preliminary mass spectroscopy studies show
that quercetin is not in the aqueous fraction of the extract
(unpublished observations, PMM). Additionally, the
level of retinoblastoma phosphorylation was decreased
following exposure to rosehip extracts, demonstrating the
cell cycle inhibitory capacity of these extracts. Decreased
retinoblastoma phosphorylation has previously been ob-
served in C6 glioma cells following exposure to caffeic
acid phenethyl ester (CAPE) and natural product used by
honey bees to construct their hives [39].
We extended our studies to investigate whether rose-
hip extracts could augment the efficacy of temozolomide,
a chemotherapeutic agent used in the treatment of glio-
mas. Recent reports have shown that temozolomide
killed brain tumor cells with greater efficiency when used
in combination with epigallocatechin gallate (EGCG), a
component of green tea or quercetin [12,40]. However, in
our studies, rosehip extracts consistently demonstrated a
greater capacity to reduce cell prolifera- tion than temo-
zolomide (Figure 10). Temozolomide in combination
Copyright © 2012 SciRes. JCT
Effect of Rosehip (Rosa canina) Extracts on Human Brain Tumor Cell Proliferation and Apoptosis
544
with rosehip extracts did not synergize and promote a
greater reduction in cell proliferation (Figure 10).
Our studies indicate that rosehip extracts are able pre-
vent cell proliferation in human glioblastoma cell lines
without promoting apoptosis. The decreased cell prolif-
eration observed following exposure to rosehip extracts
correlates with the decreased phosphorylation of key
protein kinases and proteins in both the AKT and MAPK
cell proliferation regulatory mechanisms. Rosehip ex-
tracts prevent cell proliferation by stalling the cell cycle
at the G0/G1 cell cycle checkpoint, but do not initiate
apoptosis. Rosehip extracts also demonstrate a greater
anti-proliferative potential than temozolomide, a chemo-
therapeutic commonly used in the treatment of gliomas.
Taken together, our present data along with previous
findings demonstrate an anti-oncogenic role for rosehip
extracts in human glioblastoma cells.
5. Acknowledgements
This work was supported by the National Science Foun-
dation (NSF# 1038160, O. Idassi) and The National in-
stitutes of Health Research Initiative for Scientific En-
hancement (RISE) (NIH-NIGMS R25GM076162, J.
Carpenter). Patrick Martin was supported by the National
Institutes of Health (NIH-NCI 1P20CA138020-01 &
NIH-NIGMS 5P20MD000546-07).
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