The study examined the combined effects of ellagic acid (EA) and bevacizumab (BEV) on cadherin switch and angiogenesis in a rat C6 glioma cell line model. C6 glioma cells were treated with EA (100 μM) alone or in combination with BEV (100 ng/mL) for 24, 48, and 72 hours. The combination treatment significantly reduced cell viability more than either treatment alone based on BrdU staining. EA upregulated E-cadherin and downregulated N-cadherin expression at both the gene and protein levels in a time-independent manner. The combination of EA and BEV further downregulated VEGF expression at both the gene and protein levels compared

1. 75
0884-6812/19/4103-0075/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
A
RTICLES
An Official Periodical of The International Academy of Cytology and the Italian Group of Uropathology
AQCH
ANALYTICAL and
QUANTITATIVE
CYTOPATHOLOGY and
HISTOPATHOLOGY®
OBJECTIVE: To observe the combining effect of ellagic
acid (EA) with bevacizumab (BEV) on cadherin switch
and angiogenesis of C6-glioma cell line.
STUDY DESIGN: Rat C6 glioma cells were treated
with EA at 100 µM concentration in combination with
BEV at 100 ng/mL concentration for 24, 48, and 72
hours. Cell proliferation was detected by 5-bromo-2’-
deoxyuridine (Br-dU) immunocytochemistry. Expres-
sion profiles for E-cadherin, N-cadherin, and vascular
endothelial growth factor (VEGF) proteins were deter-
mined by real-time quantitative PCR (qPCR) and their
protein levels by immunocytochemistry, subsequent to
EA treatment combined with or without BEV.
RESULTS: EA in combination with BEV conspicuous­
ly reduced the cell viability of C6-glioma cells for all
incubation times. EA upregulated the expression of
E-cadherin both at gene and protein levels in a time-
independent manner (p<0.001), regardless of the pres-
ence of BEV. Conversely, EA with and without BEV
reduced N-cadherin expression and immunoreactivity
at 48 and 72 hours (p<0.001). EA combined with BEV
treatment downregulated the expression of angiogenic
protein of VEGF (p<0.001), as well as reduced its im­
munoreactivity only at 72 hours (p<0.01).
CONCLUSION: The present study suggests a suc-
cessful therapeutic efficacy of EA in combination with
BEV, probably through inhibition of the cadherin
switch and VEGF expression. EA may be an alterna-
tive treatment of drug-resistant gliomas, in combina-
tion with BEV. (Anal Quant Cytopathol Histpathol
2019;41:75–82)
Keywords: bevacizumab; cadherins; ellagic acid;
glioblastoma; glioma; VEGF; vascular endothelial
growth factor.
Glioblastoma is one of the most common aggres-
sive and malignant tumors of the central ner-
Analytical and Quantitative Cytopathology and Histopathology®
Combining Ellagic Acid with Bevacizumab
Mediates the Cadherin Switch and
Angiogenesis in a Glioblastoma Model
Abdurrahman Çetin, M.D., and Burcu Biltekin, Ph.D.
From the Department of Neurosurgery, Gazi Yas
argil Education and Research Hospital of Health Science University, Diyarbakır, and the
Department of Histology and Embryology, Cerrahpasa Faculty of Medicine, Istanbul University–Cerrahpasa, Istanbul, Turkey.
Dr. Çetin is Neurosurgeon, Department of Neurosurgery, Gazi Yas
 argil Education and Research Hospital of Health Science University.
Dr. Biltekin is Histologist and Embryologist, Department of Histology and Embryology, Cerrahpasa Faculty of Medicine, Istanbul Uni-
versity.
Address correspondence to: Abdurrahman Çetin, M.D., Department of Neurosurgery, TR HSU Diyarbakır Gazi Yas
argil TRH, 21100
Üçkuyular, Diyarbakır, Turkey (acetin2147@gmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. vous system, with a poor prognosis in children
and adults.1 Establishing a satellite tumor group
around the primary tumor because of its uncon-
trolled aggressive behavior and invasion, the dis-
ease is pathologically defined by its enormous
vascular density and concomitant necrosis.2 Sur-
gery, radiation, and chemotherapy are useful but
severe options to manage glioblastoma; the sur-
vival duration of patients with malignant glio-
mas has been reported at between 14 weeks (no
treatment) to 40–50 weeks.1 Glioblastoma has the
high grade types designated by the World Health
Organization (WHO). Due to the aggressive na-
ture, glioblastoma cells eventually infiltrate sur-
rounding brain tissue and repopulate to form sec-
ondary lesions, resulting in approximately 100%
recurrence in local regions.3,4 Thus, the develop-
ment of a combination of innovative therapeutic
strategies is urgently required to prevent recur-
rence, prolong patient survival, and improve qual­
ity of life.5 It is vital to develop new combinations
of potent agents with basic chemotherapeutics
that are highly efficacious and safe to optimize
the efficacy of cancer treatment, resulting in a more
suitable choice for chemotherapy.
In recent years, a number of studies have
shown that the combination of phytochemical
compounds such as phenolic acids and flavonoids
extracted from fruits and vegetables can increase
the cytotoxic or antiproliferative effects of chemo-
therapeutics.5-8 A type of natural dietary polyphe-
nol compound, ellagic acid (EA) exists in several
plants and fruits6 and has been shown to have
several pharmacological properties against tu-
mors, such as anticarcinogenic effects through cell
cycle arrest, inhibition of tumor formation and
growth, and induction of apoptosis9 or by sup-
pressing angiogenesis.10 EA is also known to have
antioxidant, antifibrosis, and chemoprevention ef­
fects.11 However, its combinatory effect with com-
mon chemotherapeutics used in glioma patients is
still poorly understood.
Bevacizumab (BEV), the anti–vascular endothe­
lial growth factor (anti-VEGF) monoclonal anti-
body, is generally used in the treatment of recur-
rent gliomas with WHO grade II or III.12 Recently,
antiangiogenic therapy with BEV has shown a
high but transient efficacy in glioblastoma.13 BEV
has been known to increase intratumoral hypoxia
and glycolytic activity, resulting in elevated acidi-
fication of the microenvironment and invasiveness.
Therefore, it is suggested that therapies that tar-
get the metabolic adaptation mechanisms of cells
may provide a synergistic effect on BEV therapy.
In fact, a number of preclinical studies have indi­
cated that BEV, when combined with inhibitors
targeting invasion mechanisms and cellular me­
tabolism, reduced the tumor progression.13,14 How-
ever, its action mechanism on the cadherin switch
and combination effect with EA have not been
elucidated in gliomas yet. In order to identify the
mechanism of possible synergistic activity of EA,
we attempted to examine the combining effect of
EA with BEV on the cadherin profiles and VEGF
expression levels of C6-glioma cell line.
Materials and Methods
Cell Culture
C6 glioma cells obtained from the American Type
Culture Collection were cultured at 37°C in 95%
humidified air with 5% CO2 in Dulbecco’s modi­
fied eagle medium (DMEM), supplemented with
10% fetal bovine serum, 100 U/mL penicillin, and
100 µg/mL streptomycin. Cells were subcultured
on every 3rd day using trypsin. All concentra-
tions were handled according to dose experi-
ments as a range of 1, 10, and 100 µM for EA and
1, 10, and 100 ng/mL for BEV, determined using
the doses described in the literature.15,16 The ac-
cepted doses of EA (Sigma Aldrich, E2250-10G,
St. Louis, Missouri) (100 µM) and BEV (Altu-
zan, Roche, Istanbul, Turkey) (100 ng/mL) were
added to the media and cells were incubated for
24, 48, and 72 hours. Cells were divided into
4 groups for every incubation time: the control
group, BEV group, EA group, and a combination
of BEV and EA (BEV+EA). All experiments were
repeated at least 3 times.
Br-dU Cell Proliferation Assay
Analysis of 5-bromo-2’-deoxyuridine (Br-dU) pro-
liferation was performed according to the litera-
ture, based on immunocytochemistry.17 Br-dU was
purchased from Santa Cruz Biotechnology (SC-
32323; Santa Cruz, California); the Histostain-Plus
Bulk Kit was from SensiTek ScyTek Laborato-
ries (Logan, Utah). Mouse monoclonal anti-Br-dU
(Bu20A, SC-20045; Santa Cruz Biotechnology) was
used as the primary antibody (1:200, overnight).
As a chromogen, the aminoethylcarbazole (AEC)
chromogen (SensiTek ScyTek Laboratories) was
applied. Br-dU labeling was assessed by 2 re-
searchers, and the proliferation index was calcu­
lated by evaluating at least 3,000 cells and scored
76 Analytical and Quantitative Cytopathology and Histopathology®
Çetin and Biltekin

3. as a number of positively stained cells/total num-
ber of cells counted.17
Immunocytochemistry
C6 glioma cells cultured on cover slips were in-
cubated for 24 hours; subsequently, groups of EA
and BEV were established. After 24, 48, and 72
hours of treatment, the experiments were ter-
minated and repeated 3 times. The cells were
fixed with cold methanol for 5 minutes and im-
munostained using indirect streptavidin immu-
noperoxidase method using an anti-polyvalent
Horseradish Peroxidase (HRP) Kit (SensiTek Scy-
Tek Laboratories) for detecting E-cadherin, N-
cadherin, and VEGF proteins. The cover slips
were incubated overnight at 4°C with primary
antibodies, namely anti-E-cadherin (Santa Cruz
Biotechnology; sc-7870), anti-N-cadherin (Biorbyt,
California; orb11100), and anti-VEGF (Abcam,
ab46154) antibodies diluted according to their
protocols. The antigen-antibody complex was
subsequently visualized with AEC Substrate De­
tection System (SensiTek ScyTek Laboratories). In-
tensity of immunoreactivity was evaluated semi-
quantitatively by H-SCORE analysis according to
the literature.17
Expression Analysis
Total RNA was extracted using Total RNA Puri-
fication Kit according to the manufacturer’s pro-
tocol (Jena Bioscience, Germany). cDNA was
reverse-transcribed using the SCRIPT cDNA Syn-
thesis Kit (Jena Bioscience), according to the man­
ufacturer’s instructions. Real-time quantitative
polymerase chain reaction (qPCR) was conducted
in a CFX96 Touch (Bio-Rad, USA) machine using
qPCR GreenMaster UNG (Jena Bioscience). The
primer pairs were as follows:
E-cadherin, F: 5’-CTCTACTCTCATGCTGTGTCATC-3’
and R: 5’-CTCTGGCCTGTTGTCATTCT-3’;
N-cadherin, F: 5’-GAGAGGAAGACCAGGACTATGA-3’
and R: 5’-TCTCGTCTAGCCGTCTGATT-3’;
VEGF, F: 5’-CACTTCCAGAAACACGACAAAC-3’ and
R: 5’-CTGGTCGGAACCAGAATCTTTA-3’;
GAPDH, F: 5’-GCAAGGATACTGAGAGCAAGAG-3’
and R: 5’-GGATGGAATTGTGAGGGAGATG-3’.
After normalization to levels of GAPDH, the rela-
tive amount of E-cadherin, N-cadherin, and VEGF
transcripts in treated cells compared with controls
were calculated as means±standard error of the
mean (SEM).
Statistical Analysis
Semiquantitative and quantitative data from all
groups were evaluated statistically by GraphPad
InStat version 3.06 program (GraphPad Inc., Cal-
ifornia). All data were presented as the mean±
SEM. The mean of continuous variables was com-
pared with one-way analysis of variance, and vari-
ations between groups were compared with the
Tukey-Kramer multiple comparison test. The val-
ues p<0.05, p<0.01, and p<0.001 were accepted
as statistically significant.
Results
Treatment with Ellagic Acid in Combination with
Bevacizumab Enhances Inhibition of Cell
Proliferation
To determine whether EA could potentiate the
inhibitory effects of BEV on the proliferation of
C6 glioma cells, we semiquantitatively analyzed
the effects of EA combined with or without BEV
on cell proliferation using the Br-dU proliferation
assay. Treatment with EA alone time-dependently
suppressed the cell proliferation significantly as
compared to the control groups (p<0.001) (Ta-
ble I). Moreover, combined treatment with EA and
BEV more significantly enhanced BEV-mediated
inhibition of the cell proliferation as compared with
BEV alone (p<0.001).
EA Regulates E-Cadherin and N-Cadherin Expression
Both at Gene and Protein Levels Independently of
BEV
To determine the regulatory effects of EA on the
cadherin profile of glioma cells, E-cadherin and
N-cadherin mRNA and protein levels were in-
vestigated by qPCR (Figure 1) and immunocyto-
chemistry (Figures 2–3), respectively. Independent-
ly of the treatment time points, treatment with
EA alone significantly upregulated the expression
of E-cadherin (p<0.001) and immunoreactivity of
Volume 41, Number 3/June 2019 77
Combining Ellagic Acid with Bevacizumab in a Glioblastoma Model
Table I Proliferation Indexes of C6 Glioma Cells in the Control,
EA, BEV, and Combination (EA+BEV) Groups
Time Control EA BEV EA+BEV
24 h 84.87±2.25 54.52±4.69a,b 49.42±1.47a,b 7.94±1.30a
48 h 88.48±2.37 19.75±3.99c 54.92±2.57c 6.94±0.93c
72 h 86.10±1.65 8.76±1.5a 45.78±0.31a,b 4.86±1.65a
EA = ellagic acid, BEV = bevacizumab.
ap<0.001 vs. the control group.
bp<0.001 vs. the ellagic acid+bevacizumab group.
cp<0.001 vs. all groups.

4. E-cadherin in C6 glioma cells, while EA combined
with BEV also elevated E-cadherin expressions at
hours 24 and 48 (p<0.001 and p<0.01, respective-
ly) but not at hour 72 (Figure 3).
EA treatment downregulated the gene levels of
N-cadherin dramatically at 48 and 72 hours of in-
cubation (p<0.001) regardless of the presence of
BEV, although solely BEV exposure failed to re­
-
duce N-cadherin expression under the levels of
the control group in time (Figure 3). Immunocyto­
chemistry showed that EA succeeded in decreas-
ing the protein levels of N-cadherin in comparison
to solely BEV treatment only at 72 hours, suggest­
ing a modulatory effect of EA on cadherin switch
in glioma cells in a time-dependent manner.
EA With or Without BEV Downregulates the
Expression of VEGF in Long-Term Exposure Both at
Gene and Protein Levels
As a clinical antiangiogenesis agent,18 BEV nor­
mally downregulated the expression of VEGF only
at the first 24 hours’ incubation on glioma cells
both at the gene and protein levels with a high
significance (p<0.001) but then became nonre­
sponsive at the later hours (Figure 4). In contrast,
EA succeeded at reducing the expression and pro-
tein levels of VEGF alone dramatically at 48 hours
(p<0.001 and p<0.05, respectively) and 72 hours
(p<0.001). In combination with BEV, EA downreg-
ulated VEGF expression (p<0.001) and decreased
its immunoreactivity (p<0.01) only at 72 hours
78 Analytical and Quantitative Cytopathology and Histopathology®
Çetin and Biltekin
Figure 1
A graphical presentation of
E-cadherin and N-cadherin
expression results of ellagic
acid (EA) and bevacizumab
(BEV) treatment on C6 glioma
cells. *p<0.05, **p<0.01, and
***p<0.001 vs. all groups.
Figure 2 Microphotographs and graphical presentation of E-cadherin immunocytochemical results of ellagic acid (EA) and bevacizumab
(BEV) treatment on C6 glioma cells. Magnification: ×400. **p<0.01 and ***p<0.001 vs. control group.

5. (Figures 4–5), suggesting a potentiating effect of
EA on BEV for inhibition of angiogenesis in long-
term exposure.
Discussion
In an attempt to treat glioblastoma, recent re-
search has focused on combining phytochemical
polymers or compounds with current chemother-
apeutics that do not affect overall survival of
glioma patients.19-21 These combined approaches
may be more advantageous over the traditional
single chemotherapy regimens for application in
aggressive types of tumors, including brain tu-
mors.22 In the present study the effects of com-
bined EA (a naturally occurring dietary polyphe-
nolic compound23) and BEV (an antiangiogenic
chemotherapeutic22) therapy were investigated
on the expression of E-cadherin and N-cadherin,
which are epithelial-to-mesenchymal transition
(EMT) markers, and of VEGF, a common angio-
genic marker, in an in vitro model of C6 glioma
cells. We demonstrated that EA treatment with
or without BEV elevated selectively the expres-
sion of E-cadherin and reduced N-cadherin ex-
pression in glioma cells at the mRNA and protein
levels. However, long-term application of EA with
BEV decreased the expression of angiogenic pro-
tein of VEGF at gene and protein levels.
The EMT promotes the migratory and invasive
capabilities of tumor cells without a loss in the
proliferation and viability capacities.24 The signals
altering the EMT process in several cancers may
also induce the mesenchymal features of severe
gliomas. Additionally, the EMT process is a prom-
inent activator of the cancer stem cell pheno-
type.25 Neural stem cell markers are indicated to
be expressed in the mesenchymal subtype of glio-
blastoma and is related to an aggressive pheno-
type.26 In vitro studies reported that the glioma
Volume 41, Number 3/June 2019 79
Combining Ellagic Acid with Bevacizumab in a Glioblastoma Model
Figure 3 Microphotographs and graphical presentation of N-cadherin immunocytochemical results of ellagic acid (EA) and bevacizumab
(BEV) treatment on C6 glioma cells. Magnification: ×400. *p<0.05, **p<0.01, and ***p<0.001 vs. control group.
Figure 4 A graphical presentation of VEGF expression results of
ellagic acid (EA) and bevacizumab (BEV) treatment on C6 glioma
cells. ***p<0.001 vs. all groups.

6. cells expressing these stem cell markers are highly
invasive to neighboring tissues and resistant to
chemo- and radiotherapy.27,28 The various down-
stream pathways of the EMT essentially involve
an E-cadherin to N-cadherin shift, and both of
these molecules are pertinent to the cancer mech­
anisms of invasion and metastasis and to the
therapeutic resistance of gliomas. This cadherin
switch is a possible therapeutic target of com-
bined therapies as in other incurable cancers.29
Noh et al evaluated the expression of E-cadherin
and N-cadherin with respect to the survival rates
of a large series of glioma patients and reported
that the EMT process in gliomas may be exacer-
bated by elevated levels of N-cadherin expres-
sion, resulting in adverse prognostic outcomes.30
In the present study, EA significantly reversed the
switch of E-cadherin to N-cadherin by downreg-
ulating N-cadherin and upregulating E-cadherin
expression, suggesting an antiproliferative and
cytotoxic effect of EA by interfering in the cad-
herin shift in glioma cells. EA combined with BEV
may also exert a similar effect on cells but not as
that reported for sole EA treatment.
Glioblastomas with the mesenchymal features
are associated with a shorter overall period and
progression-free survival periods and with severe
aggressiveness of cancer, showing a therapeutic
resistance against traditional radiotherapy and
chemotherapy.11 In fact, the clinical reflection of
invasive features is obviously induced by EMT.
A study by Kahlert et al suggested that a small
subgroup of glioma cells undergoes the molecu-
lar processes that are related to the cytoskeletal
reorganization and resistance to apoptosis. Hence,
the tumor cells become excessively motile and in-
vasive and shortly thereafter gain the treatment-
resistant features.29 To overcome this resistance
in gliomas, we suggest promoting the chemo­
therapeutic effects of BEV by combining it with
EA, which can be safely combined with drugs,
especially for aggressive and persistent cancers.
As glioma cells undergoing the EMT gain the
potential to induce metastasis and invasion, the
cancer progression seems to involve upregulated
N-cadherin expression, resulting in therapeutic
resistance. In contrast, the microenvironment of
the tumor, including hypoxic conditions and mi-
crovascular proliferation, highly influences the
EMT process. Tissue hypoxia is known to direct-
ly induce the EMT and VEGF expression in the
migrating cells of the tissues.31,32 Until now, BEV
in combination with EA has not been shown in
gliomas yet. Thus, we maximized the efficacy of
an anti‑VEGF therapy with EA and suggest an
inhibitory role of EA on the expression profile of
VEGF in C6 glioma cells.
Taken together, long-term application of EA in
combination with BEV exhibits antiproliferative
activity in the C6 glioma cell line through alter-
80 Analytical and Quantitative Cytopathology and Histopathology®
Çetin and Biltekin
Figure 5 Microphotographs and graphical presentation of VEGF immunocytochemical results of ellagic acid (EA) and bevacizumab (BEV)
treatment on C6 glioma cells. Magnification: ×400. *p<0.05, **p<0.01, and ***p<0.001 vs. control group.

7. ing VEGF expression and interfering in cadherin
switch, suggesting an enhancing effect of EA on
glioma cells which become unresponsive to the
BEV therapy in long-term chemotherapies. The
findings of the present study are going to open
a possibility and target for further evaluation of
combined therapy of EA and BEV in a poten-
tial clinical trial for resistant and incurable brain
tumors. In the future, the data of in vivo and
clinical studies may indicate that the combina-
tional strategy exerts a much higher efficacy in
treating gliomas than single administration of
chemotherapeutic drugs, and thus it may help in
reducing the dosage and minimizing the side ef­
fects of cytotoxic therapy.
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